Louisiana State University
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LSU Historical Dissertations and Theses
Graduate School
1991
The Frost Tolerance of Tobacco Plants
Transformed With the Gene Encoding the
Antifreeze Protein From Winter Flounder.
Jung-sook Lee
Louisiana State University and Agricultural & Mechanical College
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Recommended Citation
Lee, Jung-sook, "The Frost Tolerance of Tobacco Plants Transformed With the Gene Encoding the Antifreeze Protein From Winter
Flounder." (1991). LSU Historical Dissertations and Theses. 5194.
http://digitalcommons.lsu.edu/gradschool_disstheses/5194
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T he frost toleran ce o f tob acco plan ts transform ed w ith th e gene
en co d in g th e an tifreeze-protein from w in ter flounder
Lee, Jung-Sook, Ph.D.
The Louisiana State University and Agricultural and Mechanical Col., 1991
UMI
300 N. ZeebRd.
Ann Arbor, MI 48106
THE FROST TOLERANCE OF TOBACCO PLANTS
TRANSFORMED WITH THE GENE ENCODING
THE ANTIFREEZE-PROTEIN FROM WINTER FLOUNDER
A Dissertation
Submitted to the Graduate Faculty of the
Louisiana State University
Agricultural and Mechanical College
in partial fulfilment of the
requirements for the degree of
Doctor of Philosophy
in
The Department of Biochemistry
by
Jung-Sook Lee
B.S. National Fisheries University, Pusan, Korea, 1981
M.S. Seoul National University, Suwon, Korea, 1983
August 1991
ACKNOWLEDGEMENTS
My
Je s s e
sincere
M.
thanks
Jaynes,
go
whose
to
my
ma jor
support,
professor,
Dr.
encouragement
and
patience enabled me to complete my dissertation.
My work
would not have been fruitful without his full support.
I
Chang,
would
R.A.
committee.
like
to
thanks
Laine,
E.
S.
Spe cia l
Drs.R.
Younat han
th anks
go
to
C.
for
my
Gayda,
S.
H.
ser vin g
on
my
colleagues,
Ms.
Martha Juban and Dr. Toti Nagapala.
My deepest appreciation goes to my family for their
love and support. My husbund,
Joon-Ho,
and son, Min-Sang,
and my parents in Korea have been the most valuable asset
in
pursuit
of
my
educational
goals.
The ir
hel p
has
helped me to endure all the difficult times successfully.
TABLE OF CONTENTS
page
ACKNOWLEDGMENTS
----------------------------------
ii
TABLE OF CONTENTS
----------------------------------
iii
LIST OF TABLES
----------------------------------
iv
LIST OF FIGURES
----------------------------------
v
LIST OF ABBREVIATIONS
----------------------------------
vii
ABSTRACT
viii
INTRODUCTION------------ ----------------------------------
1
LITERATURE REVIEW
----------------------------------
4
MATERIALS AND METHODS
----------------------------------
30
---------------------------------
69
RESULTS AND DISCUSSION
SUMMARY
----------------------------------
108
REFERENCES
----------------------------------
110
V ITA
----------------------------------
12 6
iii
LIST OF TABLES
page
1.
E.
coli strains
----------------------------------
2.
A g robacterium tumefaciens strains
3.
Plasmids used for cloning
4.
GUS activities of transgenic tobacco plants
5.
Kanamycin gene segregation
-----------------
32
------------------------
33
----
85
-----------------------
93
6 . Enhancement of frost tolerance of seedlings
iv
31
----- 105
LIST OF FIGURES
page
1. Sequence of mature antifreeze-protein from
winter flounder
-------------------------------------
6
2. Hydrogen bonding of the antifreeze-protein with
ice lattice prevents ice crystal growth
---------- 9
3. Schematic representation of AFP interaction
with ice
----------------------------------------------- 11
4.
Diagram of AFP synthesis in winter flounder
----- 14
5. The amino acid and nucleotide sequences of
--------------antifreeze protein cDNA clone IIA7
15
6 . Cloning of the mature region of antifreeze gene
into pBR322
------------------------------------------
39
7. Cloning of the antifreeze gene into
p B I 1 2 1 and pMON200
---------------------------------- 40
8 . Cloning of the mature AFP gene into pMON530
9.
Determination of pMON530AF orientation
----- 43
----------
44
10. Maps of the pIBI7 6AF and pCa2AF for further
cloning into plant transformation vectors
-------
45
11.
Maps of the final clones
---------------------------- 47
12.
Determination of pBI121AF orientation
13.
Determination of pMON200AF orientation
-----------
49
----------
50
14.
Agarose gel showing the presence of 174-bp
BamHl fragment of AFP gene from pBR322AF
-------- 7 0
15.
Agarose gel showing the presence of 324-bp
EcoRV-EcoRl fragment of pMON530AF clones
--------
16.
Agarose gel showing the orientation of pMON530AF —
v
71
72
17.
Agarose gel
showing the presence of 1.1-kb
Hindlll fragment of pBI12lAF and pMON200AF
------
74
18.
Agarose gel
showing the orientation of pBI121AF
—
75
19.
Agarose gel
showing the orientation of pMON200AF —
76
20.
Southern hybridization analysis of plasmid DNAs ----
21. Southern hybridization of transformed
A. tumefaciens LBA4404/pBI121AF
---------22.
23.
79
Southern hybridization analysis of A. tumefaciens
GV3111SE transformed with pMON200AF
-------------Leaf disc transformatin
77
80
----------------------------- 81
24. Tobacco leaf disc transformation with
A. tumefaciens GV3111SE/pMON2 00AF
----------------
82
25.
-------------------------
83
26. Southern hybridization analysis of tobacco plants
transformed with LBA4 404/pBI121AF
----------------
88
27. Southern hybridization analysis of tobacco plants
transformed with GV3111SE/pMON200AF
--------------
89
28. Southern hybridization analysis of tobacco
-------------first generation progeny plant DNAs
92
Transgenic tobacco plants
29. Northern hybridization analysis of total RNA from
tobacco plants transformed with LBA4404/pBI121AF -- 96
30. Northern hybridization analysis of total RNA from
tobacco plants transformed with GV3111SE/pMON200AF- 97
31. Western blot analysis of total protein from
----------------------------------transgenic plants
100
32. Silver staining of total protein from transgenic
plants
------------------------------------------------- 103
33.
Comparison of freezing tested tobacco plants
vi
---
106
LIST OF ABBREVIATIONS
AFP
Antifreeze-protein
AFGP
Antifreeze-glycoprotein
CaMV 35S
Cauliflower mosaic virus 35 S promoter
Ti plasmid
Tumor inducing plasmid
LIH
Left inside homology
NOS3 1
Nopaline synthase 3' polyadenylation signal
NPT II
Neomycin phosphotransferase II
GUS
P~Glucuronidase
MS salts
Murashige and Skoog salts
MUG
4-methyl umbelliferyl p-D-glucuronide
MU
4-methylumbelliferone
X-gluc
5-bromo-4-chloro-3-indol-l glucuronide
BA
Benzyladenine
NAA
Napthaleneacetic acid
2,4 D
2,4 dichloro-phenoxyacetic acid
CAP
Calf alkaline phosphatase
MES
4-morpholine ethansulfonic acid
MOPS
Morpholinopropane sulfonic acid
PMSF
Phenylmethyl sulfonyl fluoride
AP
Alkaline phosphatase
BCIP
5-bromo-4-chloro-3-indoly1-phosphate
NBT
Nitro blue tetrazolin
vii
Abstract
The
can
winter
survive
producing
flounder,
Pseudopleuronectes
in seawater at temperatures
antifreeze-proteins
freezing point
of
cellular
(IIA7
which
is p r o ces sed to a mature protein
gene
in clu din g
which
the
encoding
a
start
allo we d
promoter.
a
o nly
91
the
which
amino
mature
methionine,
e nh anced
was
e xp ression
vector
pMON200
freezing by
d e p r ess
The
a ci d
the
antifreeze
preproprotein
of 53 amino acids.
ant if r e e z e - p r o t e i n ,
clon ed
from a
The AFP gene construct was
intermediate
pBI121.
enco de s
be l o w
fluids.
gene
This
cDNA)
their
(AFPs)
americanus,
and
into
a p l a sm id
double
C aM V
35S
subcloned into both
the
binary
vector
After triparental mating and infection of tobacco
leaf discs with Agrobacterium tumefaciens containing either
pB I1 2lA F
or pMON200AF,
transgenic plantlets
were
which were kanamycin resistant and GUS positive.
analysis
confirmed
integration.
gene were
The
the
presence
tra nscription
of
levels
obtained
Southern
single-copy
of
the
gene
antifreeze
significantly higher than those of a single CaMV
35S promoter and a Western blot confirmed the synthesis of
the
immunoreactive
5.5
kd
transgenic tobacco tissues.
v/iii
antifreeze-protein
Several
individual
in
the
plant
seedlings which were kanamycin resistant were selected and
tested for frost tolerance.
pl ants
s ur vi v e d
confirmed
the
than
the
ability
of
At
least
control
this
30% more transgenic
plants.
fish
increased frost-tolerance to plants.
These
protein
to
results
co nfer
Introduction
Plants may experience different environmental stresses
such as freezing,
stresses
is
cells.
The
de hydration
hours
or
acquire
l owering
maj or
of
by
in
freezing
When plants
fr eez in g
the plant
l e a di ng
t he
p o t e nt ial s
caused
cold acclimation
parameters
Common to these
are exposed
tolerance.
an
expression
a
new
cellular
for a few
can
process
During
is
cold
several physiological
improved
of
plant
plants
This
or cold hardening.
cells m o di fy
to
the
is
low no nfreezing temperatures,
increa se d
acclimation,
water
stress
in plants.
days to
known as
induce
a
drought and salt stress.
c ol d
set
resistance
of
genes.
and
Cold
acclimation is the primary adaptation to freezing stress in
the
plant
system.
C old
accli ma tio n
studied to elucidate
the
as
mechanism
to
reveal
tolerance.
the
Ho wever,
has
been
extensively
cold acclimation process
the
acclimation is not yet clear.
of
freezing
molecular
injury
basis
cont e n t ,
in
free
su g a r
abscisic
composition,
but
acid
cause
concentration,
con te nt,
and effect
be established.
1
for
and
cold
Many biochemical alterations
have been shown to occur during cold acclimation,
changes
as well
and
including
soluble
protein
membrane
relationships
l ipi d
remain to
Another attempt to increase frost tolerance
will
be
the
originating
application
fr o m
fishes.
of
in plants
antifreeze-protein
Because
the
AFP
(AFP)
from
winter
flounder exerts a profound antifreeze effect by depressing
the freezing point of fish's body fluids,
of the AFP
gene
into a plant
the
system m a y be
introduction
a challenging
area to increase the frost tolerance in plants.
Exposure of plant tissues in a solution of the winter
flounder antifreeze-protein has confirmed the effectiveness
of the AFP in decreasing the freezing temperature of plant
tissues.
In addition,
one of the
cold-induced proteins,
the kinl gene product expressed in cytoplasm,
composition to that of winter flounder AFP.
indicates
that
the plant
in response to cold stress.
sugg es t
that
cytop la smi c
it may be
the
a natural
expression
location
is
likely
of
to
has a similar
The similarity
a n t ifr eez e- pro te in
the
The above
AFP
confer
of
findings
gene
w ith in
increased
a
frost
tolerance in plants.
Therefore,
introduction
plants
to
the
purpose
of the winter
obtain
fros t
of
t hi s
flounder AFP
tolerance.
research
gene
is
the
into tobacco
Because
s u dd en
temperature changes can occur in early spring or late fall
that do not allow sufficient time for plants to become cold
acclimated,
plants incure severe cold damage.
If plants
contained
the
AFP
gene,
which
expressed constitutively,
diminished,
will
be
designed
to
be
then this freezing injury will be
and the plants
will have time to achieve cold
acclimation.
In this dissertation,
confer
which
frost
was
tolerance
we report the ability of AFP to
in plants.
The
designed
to
c o n ta in
its
codons,
was
p l ace d
u nd er
termination
duplicated cauliflower mosaic virus
enhanced
expre ssi on .
introduced
into
The
tobacco
Agrobacterium-mediated
stable
tobacco
in tegration
plant
expression
was
and
plant
own
the
(CaMV)
gene
determined.
control
the
of
35S promoter for
by
the
Finally,
and their
gene,
and
t r an sfo rma ti on
of
AFP
initiation
con s t r u c t
plants
inheritance
in these plants
investigated.
AFP
mature
frost
was
then
using
an
system.
AFP
the
The
gene
into
AFP
gene
resistance was
4
Literature
1. Antifreeze-Protein
Proteins
acting
review
(AFP)
as a antifreeze
substance were
found
in studies of fishes in the polar oceans and the near shore
waters
of the north temperate
season,
oceans.
these oceans are at the freezing point of seawater
(-1.9°C),
a temperature well below the freezing point of a
typical marine teleost
(-0.8°C),
can
s eaw a t e r
survive
in
temperate marine
that
fishes,
electrolyte present
the
observed
The
r e ma ini ng
f r eez ing
f re ezing
glycoproteins
al.,1984;
sodium chloride
point
but
In
most
is the principal
in fishes
dep r e s s i o n
substances,
depression,
and
temperature.
inhabiting
it is responsible for only 40-50% of
antifreeze
point
but fishes in these oceans
in the blood,
freezing environments,
were
proteins
(DeVries, 1984) .
responsible
identified
( D e V r i e s ,1988;
as
for
a
the
set
of
Burcham
et
O ’Grady et al.,1982; D eVr i e s , 1983).
Antifreeze-glycoproteins
ranging between
repeat
During the winter
2.6 and 34 kd.
( A l a - A l a - T h r >n
(-Naga-Gal)
a l . , 1970) .
(AFGPs)
with
have molecular masses
They contain a tripeptide
a
attached to the threonyl
disaccharide
residues
moiety
(DeVries et
Antifreeze-peptides
kd
are
of
th r e e
H e w , 1990).
a-helical
types
Type
AFP
sculpins.
1
Type
2
kd)
et
al ani ne -ri ch ,
found
the
al.,1988;
in
Type 3 are the AFP
and
sequence.
is the most
The
flounders
AFP
(Mr
14
6-7 kd)
(Mr
extensively
typ e
1
and
amp hiphilic,
w i nt er
cys ti n e - r i c h
D a vie s
which lacks any distinctive features
composition
flounder
the
are
found in sea raven.
ranging between 3.3 and 14
(Davies
are
3-5
(Mr
in eel pouts,
(AFPs)
AFP
and
kd)
found
in its
of
w int er
characterized AFP
in its
structure and mechanism.
1) Mechanisms of AFP action
Many
researchers
mecha ni sm
its effective
variations
th e
antifreezing
(Pseudopleuronectes americanus)
action and its small
size.
from
because of
There are
some
in the size and amino acid composition of AFPs
depending on the
fish's
Gourlie
studied
and the structural requirements of the AFP
winter flounder
from
have
et
serum
separation method used to purify the AFP
(Scott
al. , 1 9 8 4 ;
et
al.,1987;
Pickett
et
Scott
al. , 1 9 8 4 ;
et
al.,1985;
Davies
et
a l . ,1984).
The
between
winter
3300-5000
sequences
flounder
Da.
(37 amino acids)
AFPs
The
are
have
smallest
shown
of
molecular
mass
antifreeze-protein
in figure 1.
The
6
NH2-Asp--Thr -Ala-Ser-Asp ■Ala-Ala-Ala-Ala-Ala-Ala-Ala
12
Thr -Ala-Ala- Asn ■Ala-Lys-Ala-Ala-Ala-Glu-Leu
23
(Ala)
(Lys)
Thr -Ala-Ala Asn Ala-Ala-Ala-Ala-Ala-Ala-Ala
34
(Asp)
Thr-Ala-Arg-C0NH2
Figure
1. Sequence of mature an tifreeze-protein from
winter flounder.
The protein is displayed to emphasize
the
acid
repeats
the
right-hand
11- amino
Residues
on
b egi n n i n g
wi t h threonine.
sid e
a re
numbered.
Hydrophilic residues p ost ulated to interact with the
ice
l a t t ice
the
AFP
et
a l .,
are
compo nen t
1987),
2 2 , and
A
and
26
boxed.
from
The
w i nt er
upper
flounder
residues
in pa ren th ese s
are
substitutions
the
flounder antifreeze peptide 3
sequence
se r u m
(Scott
below
that
is
positi ons
o ccu r
in
(Lin and Gross 1981).
18,
winter
7
distinctive feature is the repeating 11-amino acid sequence
of Thr-(X)2~polar amino acid-(X)7 , where X is predominantly
alanine.
The AFP
has
an CX-helical
content
over
80
% and
shown to be a rod shaped molecules.
The study of chemically synthesized AFP analogs showed
the
st ruc tu re- fun ct ion
AFP
(Chakrabartty et
aspartic
acid
formation.
stabilize
dipole
is
relationships
al.,1989a).
important
the
and
a - h eli ca l
hyd rop h o b i c
showed
activity.
are
flounder
The N-terminal
to
stabilize
charged
the
helix
The charged amino acids are ideally located to
conformation
interactions
stabilizing the AFP helix.
acids
in a winter
stable helix
Therefore,
required
for
the
of this
are
also
AFP.
Helix
inv olved
in
Two repeated units of 11 amino
formation but
a m i n i m u m of
antifreeze
lacked antifreeze
three
action
repeating
units
(Chakrabartty
et
a l . ,1 9 8 9 b ) .
AFPs
cry sta l
growth
so-ca lle d
va lu es
depress
greater
indicates
most
than
freezing
w i t h ou t
thermal
for
the
ch an g i n g
hysteresis
fish
1°C.
point
AFPs
A
by
the
melting
effect.
approach
high
a
ice
point,
Thermal
thermal
high antifreeze activity.
preventing
the
hysteresis
plateau
value
hysteresis
According
of
value
to
the
adsorption-inhibition
(DeVries, 1984;
theory
in
antifreeze
D e V r i e s , 1988) , the
4.5
A
mechanisms
dis tance
between
threonine and the polar amino acids is important because it
is the same as the distance
in an
AFP
between
ice crystal lattice
binds
to
the
hydrogen bonds
threonine
and
ice
the
(Figure 2).
crystal
between
adjacent oxygen atoms
The helical form of
lattice by the
formation
of
the side-chain hydroxyl groups of
carboxyl
groups
of
as partic
acid
with
oxygen a t o m s .
The
crystal
interaction
of
s tr uct ur e
the
AFP
important to orient
growth
direction
H e w , 1988).
studies
dipole
also
and
the
of
The AFP
hy dr o g e n
wa t e r
ice) (Yang
dipole may
et
bonds
to
the
that
the
d ip ole
the AFP to the pr i s m face
Yang
and
induce the arrangement
ice
In this model,
lattice,
is
(the normal
al., 1988;
the water dipole to which it binds.
he li x
s ho wed
of
the AFP
in ducing
water
molecules on the ice surface to align their dipoles to the
AFP
helix
dipole.
This
ordering
may
direct
furt he r
hydrogen bonding of AFP to the prism faces.
AFP
chains,
has
a m phi pat hi c
character.
Hydrophilic
are placed on one side of the helix,
o x ygen
atoms
groups
(mostly alanines)
the AFP helix.
by
hyd rogen
bonding,
and
side
which bind to
bulk y
h y d r o ph obi c
are projected on the other side of
Therefore,
once the hydrophilic side of the
9
/ \ /
Antifreeze
Peptide
S
r
Hydrogen
Bond
Figure 2. Hydrogen bonding of the antifreeze-protein with ice
lattice prevents ice crystal growth. Black circles indicate
oxygen atoms in the ice lattice that participate in hydrogen
bond formation (DeVries 1984).
10
AFP
is
bound
hydro ph obi c
crystal,
to
the
groups
prism
repel
face
of
the
the bind in g
resulting in the
ice
crystal,
of water
the
to the
ice
inhibition of ice crystal growth
along the a-axis direction
(Figure 3).
Once the AFP is bound to the pr is m face, growth of the
ice
crystal
plan e
in
the
(c-axis)
shaped
growth
structure
However,
a-direction
of
is
ice
not,
is
resulting
crystals
at high concentrations,
inhibited,
in
(Raymond
but
the
et
basal
needle
al.,1989).
the AFP may interact with
the basal plane by hydrogen bonding and inhibit the growth
of ice crystals in the c-direction as well.
suggested that b loc ki ng of growth
may
occur
plane
by
i n h i b iti ng
surface
in the
Another report
c-axis
n u c l ea tio n
on
direction
the
basal
(Harrison et al.,1987).
The
mechanism
for
su mm arized
as:
After
dipoles
the
ice
to
AFP
action,
alignment
surface,
the
of
ther ef ore ,
the
AFP
h ydrogen
can
heli x
and
be
ice
b o n d i n g between
the AFP and ice inhibits the growth of the ice crystal,
which
the
a m p h i p hil ici ty
other water
of the
AFP
contributes
to
in
deter
molecules from joining the ice lattice.
2) AFP gene structure
The
AFP
genes
of
the
w in ter
multigene family of about 40 members
flounder
exist
(Davies et al.,
as
a
1984;
11
c
A
Basal plane
a
AFP
Prism face
AFP binds preferentially to prism faces, through
dipolar and hydrogen bond interaction.
/
\\
\
\
\\\\
Continued ice growth on basal plane and continued binding of
AFP to prism faces results in bipyramidal ice crystal.
Figure 3. Schematic representation of AFP interaction with
ice.
Ice crystal growth along the a-axis direction (prism
face) is inhibited, but ice still grows on basal plane
(c-direction).
12
Gourlie et al.,1984;
Hew et al.,1988).
maps
of
genomic
wi nt er
flounder
direct
tandem
Scott et al.,1985;
Genomic Southern blots
clones
exist
showed
in
7-
repeats.
(approximately
Scott et al.,1988;
40
kb)
that
to
Five
are
the
8- kb
or
AFP
genes
ele ments
more
linked
and restriction
AFP
highly
ele me nts
wh i c h
homologous,
they
contain
show
a
some
the
in
elements
representing
clusters of AFP gene elements in the genome.
repeated
linked
gene
together,
of
Although the
single
AFP
diff er ent
gene
are
re str ict io n
patterns.
Therefore,
the cDNA clones characterized b y different
re se arc h
groups
size
amino
and
acid
and G r o s s , 1981).
clones
were
(1982);
IIA7,
showed
var iat ion s
co mposition
in
their
et
al.,1982;
(Davies
number,
Lin
Several nucleotide sequences of AFP cDNA
pr esented:
IIC10,
some
CT5,
determined
determined
by
Lin
determined by Gourlie et al
a nd
by
Davies
Gross
et
(1981);
al
and
(1984).
The synthesis and degradation of antifreeze-protein is
controlled
ph o t o p e r i o d
by
h orm ona l
response
(Hew et al.,1986).
abundant during the winter season
levels are
0.5% of the total
<0.001%
in the
of AFP,
is
summer.
synthesized
to
b oth
The AFPs
temperature
are exceedingly
(9 m g / m l ) .
The AFP mRNA
liver RNA in the winter,
The preproprotein,
and
processed in
and
but
the precursor
the fish liver,
13
secreted in the serum as a form of proprotein,
p r o ces se d as m ature
antifreeze-protein
and finally
(Figure
4).
Among
the post-translational modifications are the removal of the
glycine residue and the amidation of the c - t e r m i n u s .
The nucleotide sequence and the amino acid sequence of
IIA7
cDNA clone which
amino acids are
(72% alanine
hig h G.C
shown
content
(79%)
is
of 53 amino
acids.
The
signal
sequence
proline-rich
in Figure
four
repeating units
5.
High alanine
in the mature protein)
p r e dic te d protein
protein
contains
in the
nucleotide
91 amino acids,
acids
aa
consists
of
of 38 amino
the
pro-region
is
Only the mature form of AFP is
the
fluid.
The
activity and
The
hydrophobic-rich
in fish serum to depress the
antifreeze
sequence.
a
circulated
body
content
composed of a mature
l o n g ) , while
(17 aa l o n g ) .
11
contributes to
and a prepro -r egi on
pre-region
(21
of AFP
of
proprotein
the
reason
freezing point
itself
of
contains
for pro ce ssi ng to the
mature form is not known.
2.Freezing
Freezing
involves
the
injury
of
plant
in p l a n t s .
tissues
redistribution
of
a nd
cell
the
suspensions
liquid
water.
14
GENE
PRE
MATURE
PRO
.............................
» \*y.
if
*
r-
'>.* S ,
''
(9 kd)
Synthesis of preproprotein in the liver
PRO
MATURE
(6 kd)
«•,^
<• ; s,
j
Secretion of proprotein in the serum
MATURE
(4 kd)
Final processing to mature protein in the serum
Figure 4. Diagram of AFP synthesis in winter flounder.
Approximate molecular weight of the proteins is in the
parentheses.
15
PRE
Met Ala Leu Ser Leu Phe Thr Val Gly Gin Leu lie
PRO
Phe Leu Phe Trp Thr Met Arg lie Thr Glu Ala Asn Pro
Asp Pro Ala Ala Lys Ala Val Pro Ala Ala Ala Ala Pro
MATURE
Asp Thr Ala Ser Asp Ala Ala Ala Ala Ala Ala
Ala
12
GAC ACC GCC TCT GAT GCC GCC GCA GCA GCC GCC GCC
Thr Ala Ala Thr Ala Ala Ala Ala Ala Ala
Ala
23
ACC GCA GCC ACC GCC GCC GCA GCA GCC GCC GCC
Thr Ala Ala Thr Ala Ala Lys Ala Ala Ala
Leu
34
ACC GCA GCT ACC GCT GCC A AA GCC GCA GCC CTC
Thr Ala Ala Asn Ala Ala Ala Ala Ala Ala
Ala
45
ACC GCC GCC AAC GCC GCC GCC GCC GCA GCA GCC
Thr Ala Ala Ala Ala Ala Arg Gly Term
ACC GCC GCC GCA GCC GCC AG A GGT TAA
Figure 5.
The amino acid and nucleotide
of antifreeze protein cDNA clone
rich
region,
protein
are
respectively.
the
proline-rich
indicated
by
The numbers
IIA7.
The hydrophobic-
region,
PRE,
sequences
PRO,
an d
the
and
matur e
MA TUR E,
correspond to the amino acid
sequence of the mature protein.
16
The
location of
ice
formation may be
either extracellular
or intracellular and is strongly influenced by the cooling
rate.
by
At
relatively
intracel lul ar
the
plant
cell
ice
fast
cooling rates,
formation.
me mb ra ne s
with
The
injury is caused
ph ysi cal
g ro wi ng
contact
in tra ce ll ul ar
of
ice
causes mechanical freezing damage.
During
gradient
cr y sta l
water
extracellular
is
established
and
the
water
cell
the
to
dehydration
between
i n t r a c el lu la r
potential
cel lu lar
freezing,
of
will
ice
as
cause
the
liquid
ice,
(Steponkus,1984).
water
potential
extracellular
li qui d
compared
extracellular
a
water.
to
water
The
that
to
of
move
resulting
ice
lower
liquid
from
in
the
cellular
This dehydration results
in
a concentration of intracellular and extracellular solutes
which is called
'solution effects'.
include
volumetric
an d area
i n tr a-
and
because
of different
extracellular
contraction,
solutes,
solubilities
eutectic crystallization,
injury
damage
because
it
to
the
possible
pH
of b uf fe rin g
of
ch an ge
compounds,
effects
continued
(Mazur,1969).
p las ma
semipermeable characteristics.
equilibrium after
co nce nt rat io n
and possibly the removal of water
of hydration from macromolecules
Fre ez ing
The solution effects
the
mem br an e
plasma
is
the
major
membrane's
Whether the cell achieves
dehydration or
intracellular
17
ice
formation
is
ultimately
a
functioning of the plasma membrane
et
al., 1987).
The
plant
cell
consequence
of
(Steponkus,1984;
wall
also
the
Hincha
influences
the
severity of freezing injury, because the cell wall provides
a
protective
role
a ga in st
during freeze-thaw stress
mechanical
s tr ain s
occuring
(Bartolo et al.,1987).
1) Cold acclimation
When plants are exposed in subzero temperatures,
be c o m e
to le r an t
to
fr eez in g
temperature
they
(L ev it t, 1980) .
This phenomenon known as cold acclimation or cold hardening
is a complex response involving a variety of physiological
and
b io ch em ic al
changes
occur
content,
pr o te in
Th es e
changes.
D uri ng
in membrane
osmotic
quality
changes
lipid
se em
quantit y
to
adapt
acclimation,
composition,
concentration,
and
cold
hormonal
(Sakai
the
and
many
carbohydrate
balance
and
L a r c h e r ,1987).
p la nt s
to
ch il l in g
or
freezing temperatures and to resist any subsequent freezing
temperatures.
Changes
directly
(Steponkus
simple
in membrane
to
et
sugars,
the
al
lipid composition
freezing
1988) .
as
have cryoprotective
well
tolerance
In addition,
as
effects
certain
of
can
contribute
plant
prol ine
soluble
and
cell s
many
polypeptides,
(Van Swaaij et al.,1986;
Chen
18
et al.,1983).
The concentration of such cryoprotectants is
increased during cold acclimation.
There
are many
gene expression,
et
proteins
et
al.,19 88;
H a s k e l l , 1987;
describe
during cold acclimation
(Mohapatra
Gilmour
that
al. ,1 98 7;
changes
in
Meza-Basso
Robertson
during
co l d
et
in several plant
Kurkela
et
al.,1988).
acclimation
et
al.,19 88;
al.,1986;
The
are
probably at the transcriptional level.
changes
the
including the synthesis of new transcripts
and polypeptides,
species
reports
Guy
and
ch an ge s
regulated
in
mo s t
These cold-induced
in protein synthesis and mR N A accumulation involve
both the suppression as well as the induction of genes. For
example,
several
in cr eas ed
R u bi sc o
the
in
cold-induced
rice
synthesis
nu cl ea r
leaves
after
and other
en co de d
pr o t e i n s
are
However,
c hlo rop la st- en cod ed mRNA,
ch lorophyll
fu nct io ns
spe ci fi ca ll y
cold treatment.
a/b
strongly repressed in the cold.
ch l o r o p l a s t
are
bi n d i n g
pr ot ei n
This indicates that
disturbed
during
cold
and
are
some
stress
(Hahn and Walbot, 1989) .
Another
in c r e a s e
in
change
abscisic
J a n s s o n , 1980;
Chen
major
Daie
an d
and Gusta, 1983) .
occurring
plant
acid
in
co l d
acclimation
concentrations
C a m p b e l l , 1981;
Abscicic
hormone,
has
acid
been
Chen
(ABA),
shown
is
an
(Bornman
and
et
al.,1983;
a natur all y
to
play
an
19
important role in plant water balance and in the adaptation
of plants
to
stressful
environments.
been pr op os ed as a common mediator
stresses.
Therefore,
for plant
ABA
has
responses
to
Cold acclimation -induced freezing tolerance is
med iated by increased levels of endogeneous A B A which acts
by
activating
the
genetic
fr ee zin g-tolerance
s ys t e m
response.
responsible
ABA -in du ce d
gene
for
expression
is related to cold acclimation or freezing tolerance
and Chua,1988;
Several
genes
Lin
have
et
cDNAs
been
one,
the
the
gene
another,
relat ed
to
cold-
characte ri zed
al ., 19 90 ;
Close
et
and/or
(Kurkela
from
involved
protein
cD N A
in
the newly
r el ate d
to
cold
synthesized protein
Cattivelli
two
gene
a regulatory
F r a n c k , 1990;
and
The deduced amino
showed
co ld- ind uc ed
serves
ex p r e s s i o n
clones
ABA -in du cib le
and
a l . ,1 9 89 ;
Skriver and Mundy, 1990).
sequence
prot ei n
(Mundy
Mohapatra et al.,1988).
Bartels, 1990;
acid
the
categories
expression.
function
to
In
control
ac cli mation.
itself
of
serves
In
as a
c r yo pr ot ec ta n t.
In barley,
cold-regulated
the
proteins
deduced
genes
contain
arginine
from
cDNA
rich basic
clones
of
domains.
This suggests that these cold-induced protein may stabilize
sp ec i f i c
sequences
mRNA
are
during
cold
involved
stress
in
because
arginine-rich
protein-RNA
interactions
20
(Cattivelli and Bar te ls, 1990).
The
dehydration-induced
rec en tl y
c ha ra ct er iz ed
AB A- in d uc ed
from
proteins
several
cystein-
and
t ry pto ph an- fr ee
seedlings
units
in a conserved
and
linear order.
dehydrins are not yet known,
The
the
to
an
(Close et
contains
glycine
repeated
function of these
but the existence of a highly
conserved region with a consistent
that
were
r el ate d
Each dehydrin is extremely hydrophilic,
rich,
indicate
cDNAs
cDNA from barley and corn
al.,1989).
(dehydrins)
dehydrins
spacial arrangement may
have
a
central
role
in
gene
regulation.
As
an
example
acclimating plants,
the
po ly pe pt id es
Ar a bi do ps is
which
of
ha d
cold
Lin et al
as
cryoprotectants
(1990)
in
common
were
in
(cor)
genes
in
The distinctive
heat
They
co l d -
identified a number of
cold-regulated
hydrophilicity.
polypeptides
to
a
thaliana and wheat.
they
degree
of
stability
suggested
features
and
that
both
a high
these
COR
have a fundamental role in plants acclimating
tempe ra tur es
and
these
p o l y pe p ti de s
may
act
as
cr yo pro t ec ta nt s.
The
(1990),
kinl
gene,
c ha ra ct er iz ed
by
K ur kel a
and
Franck
is induced at cold temperature as well as by water
stress and by A B A in Arabidopsis
kinl m R N A
is
increased
thaliana.
20-fold
The level of
in cold-treated plants.
21
The
d e du ced
has
an
sequence,
sequence
amino
ac id
it
is most
of
comparison
component
of
located
protein
the
B precursor
is
deduced
the
41%.
as
amount
an
being
and
rich
in
lack of a signal
in the
cytoplasm.
Kinl
However,
p ro te in
flounder
with
the
revealed a
In addition to the small
of b io ch emi ca lly
Therefore,
hy dr oph ili c
unk nown.
from winter
certain similarity between them*.
size,
quite
Due to the
pr obably
Ki n l
is
compo sit ion ,
glycine and lysine.
fun ct io ns
AFP
Kda polype pti de
unusual
alanine,
The
6.5
similar
amino
acids
is
they suggest that Kinl protein may serve
a nt if ree ze -pr ote in
in the plant's
response
to
cold
stress.
*Alignment of the deduced Kinl protein with the AFP component B
from winter flounder.
Identical
lines,
acids
similar
amino
by
amino
acids
double
dots,
are
and
shown by vertical
a
low
degree
of
similarity by single dots.
Kinl 27 DKAKDAAAGA...GAGAQQAGKSVSDAAAGGVNFVKDKTGLNK
66
AFPB
38
1 DTASDAAAAAALTAANAAAAAKLTADNAAAAA......AATARG
In addition,
has
a small
recently been
1990).
CS7.4
cold pro tection protein,
described
represents
in E. c o l i
a
new
(Goldstein
group
CS7.4,
et
a l .,
of AFPs with no
22
similarity to the
small
fish AFPs
except
for the
size and
high proportion of hydrophilic residues.
2)
Attempts to increase frost tolerance in plants and
application of the AFP.
Attempts to improve tolerance to freezing stress have
been studied in several ways.
spring
wh e a t
was
Recently,
regenerated
fro m
freezing-tolerant
cryoselected
calli
following freezing in liquid nitrogen without the addition
of
cryoprotec tan ts
(cryoselected)
seed
(Kendall
et
al.,1990).
The
surviving
calli exhibited freezing tolerance,
progeny
of
regenerated
plants
also
and the
maintained
freezing tolerance but to a lesser degree than the
callus
itself.
ha v e
Frost
tolerance
be e n
studied
in plants
to
ba s ed
elucidate
on
the
cold
cold
mechan is m which involves osmotic adjustment,
water
potential
Guy, 1989;
Van
K u l e s z a , 1987) .
conditions,
fr eez ing
by
and
growth
Sw aa i j
Under
et
th e
the
acclimation
and changes in
capability
(Y e le nos ky
an d
a l .,
1987;
Kacperska
and
most
favorable
plants can increase tolerance
d ec r e a s i n g
ac cl ima ti on
os mo tic
hardening
to extracellular
potential
and
water
potential of the cell.
Another attempt to increase frost tolerance may be the
23
application of the AFP from winter flounder exogeneously or
endogeneously.
Exposure
of
plan t
t is su es
in
a
lmg/ml
solution of the winter flounder antifreeze protein in vitro
has revealed that AFP can function as an anti-nucleator in
plant
tissues,
average
de cr e as in g
of
1.8°C.
cry op rotectant
decrease
to
the
al.,1989).
of
These
po ss ibi li ty of
freezing
Mo r e o v e r ,
reduce
rate
the
the
ice
tem pe ra tu re
A FP
could
freezable
crystal
results
water
an
as
a
amount,
f o rm at io n
strongly
improving the
act
by
(Cutler
demonstrate
frost tolerance
and
et
the
of plants by
introduction of an AFP gene.
Certain
inter-
and
activity,
bacteria
ice
intracellularly,
and cause
Pseudomonas
promote
frost
through
damage.
s y r i n g a e var Hall
formation
ice
in
nucleation
Two bacterial
and E rw ini a
plants
species,
herbicola,
are
widely distributed on leaves of numerous plant
species,
in
which
(Lind ow
et
they
al.,1982).
antifreeze
act ivity
se rv e
th e
ice
Prody-Morreale et al
glycoproteins
of membrane
herbicola.
as
nucleators
(1988)
in h i b i t
vesicles
described that fish
t he
from the
ice-nucleating
bacterium Erwinia
These results also indicate that
can reduce the
ice formation
in other
fish AFP/AFGP
systems besides
the
fish.
Besides the in vitro application of AFP,
the AFP gene
was successfully transferee! to Atlantic salmon
al
1988)
and
al.,1987).
Drosophila
melanogaster
genome.
ex pr ess ed the AFP
of
the
The
gene,
primary
transgenic
including
a form of pr op rot ei n
provide
the
et
under the
was incorporated into a
D .m e l a n o g a s t e r
splicing and processing
transcripts.
translated and the AFP
reports
(Rancourt
The winter flounder AFP genomic DNA,
control of the heat shock promoter,
Drosophila
(Fletcher et
The
transcripts
were
was secreated into the hemolymph as
of the
winter
possibility
flounder
tha t
AFP.
Those
limited
fr eeze
resistance can be conferred to plants through the transfer
and expression of the AFP gene.
3. Agrobacterium-mediated plant transformation
Agrobacterium tumefaciens induces crown gall tumors by
transferring oncogenes from the tumor-inducing
(Ti) plasmid
to plant
involved
cells.
biosynthesis
The oncogenes encode enzymes
of
auxin
unorganized
p ro l i f e r a t i o n
segment
the
of
Ti
transm itt ed to plant
plant
n uc le ar
DNA
and
of
plasmid
cytokinin,
plant
cells.
which
is
cells and stably
(Ream,1989/
which
A
called
Binns,1988).
integration of foreign T - D N A into the plant
ca u s e
specific
T-DNA
integrated
in
is
into the
The
stable
nuclear genome
25
pro v id es
a useful
tool
transformation.
transformation
genetics
to
attack,
for A g r o b a c t e r i u m - m e d i a t e d
Th e
s ys te m
development
provides
improve
great
resistance
environmental
of
stress,
to
and
such
pot e nt ia l
plant
a
plant
in
plant
h e r b ic id es ,
bacterial
insect
an d
viral
infections.
The
begins
interaction
between
Agrobacterium
and
pl ants
with the attachment of the bacteria to the wounded
plan t
cell
surface.
Three
genetic
loci
encoded
by
the
A g r o b a ct er iu m chromosome have been defined as having roles
in A.tumefaciens attachment to plant cells:
pscA
(exoC)
(Douglas
responsible for the
a role
the
et
fibrils,
of
The ps cA
s ur f ac e
ext rac ell ul ar
0-1,2-D-glucan
locus
it se lf
compounds
chvB
are
also has
a role
polysaccharide
in
cellulose
succinylglycan,
and
substances,
and
including
that presumably help the plant
further
acting
acetosyringone
and
(Thomashow et a l ., 1987).
compounds,
against
and
polysaccharides:
Wounded plants produce numerous
phenolic
ch vA
chvB,
synthesis of 0-1,2-D-glucan which has
in attachment.
production
a l ., 1985).
chvA,
as
damage.
plant
Am o ng
several
signal
hydroxyacetosyringone,
p h en ol ic
molecules
which
expression of specific vir genes in the Ti plasmid
defend
are
induce
(Stachel
26
et al.,
1985).
exci si on
of
The
the
vir gene products are required for the
T- D N A
from
the
Ti
plasmid,
and
for
its
transfer and integration into the plant genome.
The vir region is approximately 35Kb and encodes six
transcriptional
loci:
vir G
and
(Stachel
expressed
VirA
virA,
virB,
Nester,
protein
virC,
1986) .
This interaction results
VirG pr ot ei n
by phosphorylation.
in c r e a s e s
transcription
tr ans cri pt ion
of the
A
i n t e ra ct s
compounds.
of
its
virD,
virE,
and
constitutively
with
vir
inducing
in activation of the
A c ti v at ed
VirG
protein
own
an d
induc es
virB,C ,D, an d E
gene
operons
(Stachel
and
Zambryski,
1986) . T-DNA transmission involves the action of
vir
p ro d u c t s
gene
elements,
the
cis-acting
border
seq uen ce
25 bp direct repeats that flank all T-DNA regions
from Ti plasmids.
activity
and
The virD operon encodes an endonuclease
that generates
d o u b l e -s tr a nd
cuts
specific
wi th in
the
single-st ran d nicks
b ord er
repeats.
The
and
nick
within the right border repeat initiates production of full
length single-stranded copies of the bo tto m strand of the T
region,
releases
plasmid
and
this
a
si mi la r
single-st ra nde d
(Stachel et al.,
The overdrive
with a highly
nick
(ode)
in
DN A
the
left
border
(T-strand)
repeat
from the
Ti
1987).
enhancer sequence,
conserved 6bp core,
lies
a 15bp element
near
the
right
27
border
and
transfer.
also
stimulates
production
and
T-DNA
T-strand formation and right-border nicking are
s ti mu la ted by
al .,
T-strand
1986;
the
Veluthambi
presence
et
of
al.,
overdrive
1988) .
(Peralta
A
nonspecific
single-stranded DN A bindi ng protein encoded by
and pro tects
the
single-str an ded
et
virE2 binds
T - s t r a n d s . Subsequently,
T-DNA is transfered into plant cells by a process involving
the virB o p e r o n .
T-DNA transfer is similar to the bacterial conjugation
m ec han is m
(Buchanan-Wollaston et al.,
integration
from
the
of T-DNA
or
as
into the plant
c o n j u ga ti on
variety of locations
short
tandem
1987).
mechanism.
genome
T-DNA
Agrobacterium-mediated
(direct
i nse rti on
or
at
a
as single copies
inv ert ed
into
the
likely differs
inte gr ate s
in the plant genome,
arrays
However,
the
appears to be random at the chromosomal level
repeats).
p la nt
genome
(Chyi et a l .,
1986) .
The
resultant
t r a n sf or me d
plant
cells
produce
sugar and amino acid conjugates, termed opines,
to the plant hormones auxin and cytokinin.
novel
in addition
Opines are used
by the inciting bacteria as a source of carbon and nitrogen
and as an inducer of Ti plasmid transfer between bacteria.
Efficient
tra ns mission
(transfer and
integration)
T-DNA to a plant genome does not require tumorigenesis,
of
but
requires,
trans,
in
the
al.,
the
right
25bp
b or de r
repeat
and,
in
vir genes located outside of the T region of the Ti
plasmid.
been
cis,
Therefore,
de ve lo p ed
to
introduction
adapt
of
1987/ Bevan,
1983).
the Ti plasmid-based vector system has
these
new DNAs
two
essential
into plant
1984; Fraley et al.,
features
cells
1985;
for
(Rogers
et
Barton et a l .,
The altered Ti plasmids that have had the oncogene
and opine synthase genes deleted or replaced by selectable
a n ti bi oti c
markers
and Simpson,
of
direct
been
The
referred
to
as
'disarmed'
(Shahin
1986).
As the great
lack
are
unique
cloning
size
of the disarmed Ti p l as mi d and the
restriction
into
de vel op ed for
in termediate
the
T-DNA,
whic h
sites
intermediate
introducing genes
vectors
antibiotic markers
e n d o n uc le as e
commonly
function
vecto rs
into the
contain
the
in plants
p r oh ib i t
have
Ti plasmid.
selectable
and bacteria,
the right border sequence and the multilinker site.
There
are
two
kinds
co i nt egr at ing
(cis)
vectors
Cointegrating
in ter me di at e
of
intermediate
and bi na ry
vectors
such
(trans)
as
vectors,
vectors.
p MO N2 00
must
carry DNA segments homologous to the disarmed T-DNA called
the left inside homology
(LIH)
fragment.
The LIH sequence
provides the sites to be cointegrated into the disarmed Ti
pl a smi d
by
homologous
recombination.
The cointegrated
29
intermediate vector is replicated by the Ti plasm id origin
of
replication.
B ina ry
vectors
are
develo pe d
by
finding
that the T-DNA did not have to be physically joined to the
Ti plasmid
(Hoekema et al.,
1983).
Because a binary vector
such as pBI121 has an origin of replication that
in both E.coli and A. tum efaciens,
independently of the Ti plasmid
To
introduce
A.tumefaciens,
transfection.
needs
the
the re
tw o
bacteria:
methods,
E.coli
vector
cells
E.coli
cells
vectors
co n j u g a t i o n
cells
car ry i ng
et
rapid,
carrying
to
be
the
p er
ug
However,
and
also
that
(An et al.,
mobilized
The
A.tumefaciens
r e ar ra ng em en t
method
ve ct or
a l ., 1988).
transformants
method.
the
pRK2013
he l p e r
The mobilization plasmid pRK2013 provides the RK2
in te rme dia te
direct
and
carrying a intermediate
transfer functions and the ColEl mob protein,
(Rogers
into
(triparental mating)
A.tumefaciens
disarmed Ti plasmid,
plasmid.
(An et a l ., 1988) .
The conjugation method
three
and
it is able to replicate
intermediate
are
functions
t he
low
technique
1988).
occur s
frequency by
(approximately
compared
eliminates
often
A.tumefaciens
trans for mat io n
is
DNA)
into
allowing the
is
to
the
c o n j ug at io n
re liable
much
du ri ng
of
103
th e
the
and
very
plasmid
co nj ug at ion
30
Materials and Methods
Materials
1. Bacterial Strains
E.coli
the
JM101,
JM107,
transformation
Agrobacterium
of
respectively
the
t ume fa cie ns
for the tr ans formation
HB101
and
DNA
constructs
LBA4404
of pBI121
DH5CX1 were
used
(Table
and GV3111SE
and pMON200
were
for
1) .
used
derivatives,
(Table 2).
2. Vectors
Table 3 provides a list of the plasmids used in these
experiments,
as well as the purpose of each plasmid.
3. Radioactive Compounds
[0C-32P]
purchased
and
from
England Nuclear
4.Enzymes,
[y-32P]
dN T P s
ICN Biomedicals,
(NEN)/Du Pont Co.
DNA,
(3000
Inc.
(Irvine, CA)
(Wilmington,
were
and New
DE).
RNA and Proteins
All restriction endonucleases,
T4 DNA polymerase,
Ci/ mm o le )
and the Klenow
DNA ligase,
fragment
DNA kinase,
were purchased
31
Table 1. E. coli strains
E. coli
JM101
Genotype
(lac proAB)
Reference
supE,
tra D36, pro AB,
thi/F',
lac Iqz
Messing et
al.,
(1981)
Ml 5
JM107
(lac proAB)
supE 4 4 / F '
tra D36, proAB,
HB101
hsdR17
(rk-mk+)
thi,gyrA96,endAl,
lac Iqz
M15
YanischPerron et
al., (1985)
F-,hs dS2 0( rb- mb -) ,recAl3,
Boyer et al.,
leuB6,
aral4,
(1969)
galK2,
rpsL20(str),
proA2,lacYl,
xyl-5,
mtl-1,supE44-
DH50C'
F', endAl,recAl,
lacZ M15,
hsd R ( r k- mk -) ,supE44,thi-,
gyrA96,
relAl,
(lac ZY A-a rg F), U169
BRL Focus
(1986)
8:2,9
32
Table 2. Agrobacterium tumefaciens strains
Strain
Description
Reference
LBA4404
a non-tumor-forming
Oomas et a l .,
derivative of LBA4 4 01,
(1982)
it contains a Ti plasmid,
PAL4404,
with deleted
T-region but intact
vir-region;
streptomycin
resistance
GV3111SE
disarmed strain which
carries pTi B6 S3-SE,
it contains only the TL
DN A left border sequence
and a region of homologous
Ti DNA which allows for
cointegrate formation;
chloramphenicol and
kanamycin resistance
Fraley et al.
(1985)
33
Table 3. Plasmids used for cloning
Plasmid
Size (kb)
pBR322
4.3
Purpose
initial cloning of
mature AFP gene,
Reference
Boliver
(1978)
DNA amplification
pMON 530
pIBI7 6
12.0
4.2
intermediate vector
Rogers et
for further cloning
a l . ,(1987)
cloning vector for
Dente et
use in the poly I
a l . ,(1983)
linker region
pCa2
3.3
source of the double
Kay et a l .,
cauliflower mosaic
(1987)
virus promoter
pMON200
9.5
cointegrating vector
Rogers et
compatible with
a l . , (1987)
Ag robacterium GV3111SE
pBI121
13.0
binary vector
Bevan
(1984)
compatible with
A grobacterium LBA4 404
pRK2 013
contains mob genes to
Fraley et
mobilize intermediate
al., (1985)
vectors into
A.tumefaciens during
triparental mating
34
from Bethesda Res ea r ch
New
England
B i ot ec h
Biolabs,
Corp.
Laboratories
Inc.
(Madison,
[NEB]
United
DNase,
States
WI) , P ha rm aci a
RNase,
Biochemicals
proteinase,
(Bethesda,
(Beverly,
Boehringer- Ma nnh ei m Biochemicals
and
[BRL]
[BMB]
[USB]
and DNA,
MA) ,
MD),
Pro meg a
(Piscataway,
NJ) ,
(Indianapolis,
IN)
(Cleveland,
RNA,
OH) .
and pro tei n
size
markers were purchased from BRL or U S B .
5. Chemicals
Bacto-tryptone,
bacto-yeast,
ba cto-peptone and bacto-
agar were
obtained from Difco Laboratories
Agarose,
dithiothreitol(DTT),
p u r ch as ed
from BRL
[AMERESCO]
(Solon,
bisacrylamide,
from Biorad
(Richmond,
spectinomycin,
from
Most of the
2 - m o r ph ol in e
chloride,
OH).
tetramethyl
phenol
were
and
Res ea r ch
Products
Acrylamide,
Company
N .N '- m e t h y l e n e
CA).
ampicillin,
streptomycin
Sigma
tetracycline,
and
Ch emi ca l
k a n a my ci n,
chloramphenicol
Co m pan y
(St.
Louis,
chemicals were purchased from Sigma,
ethanesulfonic
acid
(MES),
Tris- hydroxymethyl aminoethane
pyrrolidone
MI).
TEMED and ammonium persulfate were purchased
Antibiotics
obtained
or Am er ic a n
urea
(Detroit,
chloride,
sodium
MO) .
including
h ex a m i n e
cobalt
(Tris), polyvinyl
(PVP), ethylenediamine tetraacetic acid
ammonium
were
dodecyl
(EDTA),
sulfate
35
(S D S) ,
polyethylene
fluoride
gl y c o l
(PMSF), and
(P E G) ,
Sephadex
phenylmethylsulfony1
G-100.
was obtained from Morton Thiokol Inc.
Nitrocellulose
a nd
S ch ue ll
NH) .
obtained
fr o m
Stratagene
membran es
were
pu r c h a s e d
MA) .
Prepacked
Inc.
Inc.
Skoog
salts
(Lenexa,
Duralon
(La
Sephadex
UV
Jolla,
G-5 0
membranes
CA) .
were
Immobi lon
Corp.
columms
(Bradford,
ca m e
from
(Paoli, PA).
were
phytoagar and pre mixed Murashige
purchased
KS) .
The
(B A ) , n a p t h a 1 e n e a c e t i c
dichloro-phenoxyacetic
myoinositol,
(Danvers, M A ) .
from Mi lli po re
Tissue culture agar,
and
chloride
filters were purch ase d from Schleicher
(Keene,
5Prime-3Prime,
Rubidium
Haz l et on
phytohormones
acid
acid
thiamine HC1,
from
(2,4
from
Sigma.
purchased
from
Hoechst-Roussel
benzyladenine
(NAA),
D),
an d
nicotinic acid,
purchased
Biologies,
Cefotaxime
the
2,4
vit am i ns
pyridoxine were
sodium
salt
Pharmaceuticals,
was
Inc.
(Somerville, N J ) .
4-methylumbelliferone
glucuronide
glucuronide
(MUG)
(X-gluc)
Research Organics,
Inc.
and
were
(MU),
4-methyl
umbelliferyl
5-bromo-4-chloro-3-indol-l
purchased
(Cleveland,
fr om
OH).
Clontech
and
36
6 . Kits
Nick
pu rc ha se d
translation
from BMB,
and
random
BRL and USB.
Western
Promega.
blot
The
"Protoblot"
Protein
as say
kit s
wer e
The GUS assay kit
obtained from Clontech Laboratories,
The
priming
Inc.
was
(Palo Alto,
CA).
kit
was
purchased
from
kit
an d
Immunopure
Ig-G
purification kit were purchased from Pierce
(Rockford,
IL).
7. Others
The tobacco
var
Samsung)
(Department
wer e
of
University) .
and Miracle
(Jackson,
flounder
seeds
obtained
Plant
P ro -Mi x
Grow
MI) .
AFP
(Nicotians
was
tabacum var xant hii
from
Pathology,
soil,
Dr.
Jiffy pots,
antibody
a gift
Physiology and Biophysics,
from
Dr.
Flores
Pennsylvania
fertilizer were p ur ch ase d
The
Hector
raised
DeVries
and
St at e
Temik pestic id e
from BWI,
against
Inc.
winter
(Department
University of I l l i n o i s ) .
of
37
Methods
1. Plasmid construction
1) Cloning of the mature AFP gene into plasmid pBR322
The
clone,
winter
IIA7,
flounder
antifreeze-protein
which was pro vided by Dr. DeVries
of Physiology and Biophysics,
University of
digested with Hpall restriction enzyme.
fragment
was
isolated
electroel ut ion .
The
further digested
the 5'
the
with
and
SfaNl
gene.
SfaNl
th e
441-bp
fragment
Illinois),
was
by
agarose
gel
DNA
fragment
was
restriction
recognizes
enzyme
fr o m
the
to remove
sequence
(150 mM NaCl,
10 mM M g C l 2, 100 ug/ml BSA)
larger
(Department
tail and the pre-pro region of
(5/9) with the reaction buffer
pH7.5,
cDNA
The 441-bp Hpall
purified
purified
site of the poly(dG)
AFP
(AFP)
at 37°C.
the
of
GCATC
10 m M Tris-HCl
The 258-bp of
Sf a N l
digestion
was
electroeluted from the agarose gel.
The
synthetic
olig onu cl eot ide
GAC) , pr od uc in g the
BamHl
site,
linker
(GATCC
ACC
start
codon,
and the
the
first amino acid codon of the mature AFP gene,
and an nea le d with
DNA
to
90
°C
temperature.
for
the
10
258-bp DNA
m in ut es
fragment
and
then
The linker ligated DNA
was kinased
by he at in g
c oo li ng
was
ATG
to
the
room
fully digested
38
wi t h
Sa u3 A
poly(dC)
coding
the
restriction
tail
from
sequence
scheme
of
for
enzym e
the
AFP
to
gene,
the mature
the
remove
cloning
producing
AFP
of
the
gene.
the
3'
the
site
complete
Figure
m at ur e
AFP
of
6 shows
gene
into
plasmid pBR322.
The 174-bp Sau3A-digested AFP gene sequence was cloned
into
plasmid
pBR322.
pB R32 2
was
cut
treated with calf alkaline phosphatase
religation.
were
The
ligated
and
Tr a ns fo rma nts
were
tetracycline
insert
was
plasmid
AFP
DNA
se lected
by
confirmed
CAP
into
and
to prevent its
tr e a t e d
pBR322
HB101.
r es is ta nce
presence
plasmid
BamHl
E.coli
am p i ci ll in
The
by
(CAP)
and
transformed
sensitivity.
transformants.
The
m at ur e
with
of
the
and
174-bp
miniscreens
of
The resulting pl as mi d was called pBR322AF.
was
prepared
in
large
scale
q u a n ti ti es
and
purified using cesium chloride banding.
2) Construction of the expression vectors.
The plant
several
fr agment
Bglll
was
site
p M ON 53 0
(CaMV)
steps
is
35S
tr ans fo rma tio n vectors
outlined
excised
of
plasmid
loca ted
in
from
and
7.
pBR322AF
pMON530.
between
promoter
Figur e
the
were
The
a nd
The
174-bp
cloned
p ol y l i n k e r
ca u li f lo we r
the
construc ted
nopaline
BamHl
into
site
mo sa ic
in
the
of
virus
synthase
I
cut with Hpa II and 441 bp
fragment isolation
(G)10
PRE
PRO
MATURE
(Oil
HpaD
HpaD
SfaNI
Sau3AI
cut with SfaNI and larger fragment isolation
anneal and ligate the synthetic oligonucleotides
GATCC ACC ATG GAC
GTGG TACCTGTGGC
cut with Sau3AI
BamHl
pBR322
cut with BamHl
pBR322-AF
Amp
Figure 6. Cloning of the mature region of antifreeze
into pBR322.
AD
pBR322AF
4.5kb
cut with BamHl
cut with Bgin
pMON530
EcoRV
Hindm
pMON530AF
EcoRV
HinrlTTT
cut with EcoRV, Hindlll
'polylinker (Sac I)
pIBI76
4.2Kb
cut with EcoRV,
HindlB
HindEH
EcoRV
polylinker (Sac I)
pIBI76-AF
4.8kb
Amp
cut with EcoRV,SacI
I
Continued
Amp
41
EcoRV
cut with EcoRV,SacI
pCA2
SacI
3.3KB
HindDI
pCa2-AF
Amp
HindHt
4.4KB
HindDI
polylinker
(HindlH)
Amp
cut with HindHI
GUS gene
cut with HindDI
pMON200
Hindm
cut with HindDI
pIBI121
13kb
9.5kb
pMON200AF
10.6kb
pBI121AF
14.1kb
Figure 7. Cloning of the antifreeze gene into pBI121
and pMON200.
42
polyadenylation
site
(NOS
3'
terminator).
insertion of the 174-bp BamHl
of
p MO N53 0
resulting
destroyed
plasmid
both
Since
the
fragment into the Bglll site
BamHl
pMON530AF
and
Bglll
(Figure
sites
8) ,
the
in
the
plasmid
miniscreen was accomplished by EcoRl and EcoRV digestion.
Th e
orientation
restriction
EcoRV-EcoRl
digested
correct
e nz y m e
of
mapping
fragment
with
pMON530AF
of
SfaNI.
the
determined
(Figure
9) .
The
pMON530AF
was
purified
If the
orientation,
was
174-bp
digestion
134-bp and 190-bp fragments.
Otherwise,
3 24- bp
i n s er ti on
with
SfaNI
by
and
has
a
generates
it produces 280-bp
and 44-bp fragments.
The
Ca M V
35S
expres se d plant
of
the
CaM V
enhancement
activity
pro mo te r
ma tur e
p r o m o t e r is
a
tran scr ipt io nal
35S
promoter
of
plant
strong,
promoter,
sequence
ge ne s
AFP
gene
a l ., 1987) .
was
further
but
creates
with
the
Therefore,
c a r ri ed
duplication
mu c h
stronger
transcriptional
almost te n-fold higher than that
(Kay et
c on s t i t u t i v e l y
out
of the natural
cloning
of
the
p ro duc e
the
pMON530AF
was
to
duplicated CaMV 35S promoter sequence.
The
cloned
EcoRV
into
mu ltilinker
the
and
Hindl ll fragment
plasmid
region
pIBI76,
from
which
con tains
just behin d the Hindlll
is useful for further cloning
(Figure 10).
site,
a
polyl
hence
it
The EcoRV and
43
174-bp
F
•*■'■l.1■*.' ■*'■*<■
'7 ?.i|vji/ j
n s% aa«*S*%sS BSaS*%*S*%*S*ak*1
r
^
-V-V-V
fc»MV«-%»MSaSV%a%
aS aSaSiS«'
BamHl
BamHl
Bglll cut
pMON530
¥
AFP
N0S3'
35S
RK2 \
Replicoir
Nos
Nptlll
Nos
pMON530AF
322
origin
Nopalinesynthase
pTiT-37
^
Figure 8. Cloning of the mature AFP gene into pMON530.
The resulting plasmid pMON530AF was identified by the presence
of the 324-bp EcoRV-EcoRI fragment.
EcoRV
EcoRI
I*
324 bp
H
AFP
Mrr
%
'*S
11
■'V
V
.S'S*
-V
-l
i
'
V'i
SfaNI digestion
Correct
Orientation
Incorrect
Orientation
I
I
>■>«m*• \w•i*AFP
'
1
'•■S(S*S*
— 134 bp
t
»"• »i*'iV
*s*s*s»s»%
190 bp
•%•s • s*v A F P
3
r*r;r,*r;
■%
!*■I*«»
»«■
■»r_»r-r
w W *-
■■S *V
280 bp
SfaNI
Figure 9. Determination of pMON530AF orientation.
-^44
SfaNI
45
Hind ill
Sph I
Mlu I
Xho I
Apa I
Pstl
Sal I
Hinc II
Xba I
BamHl
Xma Sma
Kpn
Sac
EcoRI
EcoRV
plBI76AF
4.8 kb
Hind III
EcoRV
poly I linker
AFP
Hind III
NOS3'
35S
Sac
35S
pCa2AF
4.3 kb
Amp
Figure 10. Maps of the pIBI7 6AF and pCa2AF for further cloning
into plant transformation vectors.
46
SacI
fragment
from the
the p l a s m i d pCa2,
duplicated
pCa2AF
upstream
1111-bp
duplicated
sequence
bi na ry
the
region
Ca M V
in
CaMV
pUC
into
35S p rom o t e r with
18,
creating
p M O N 20 0
3'
cassette
This
gene which confers
so it
and
a
under
GUS
the
the
te rm ina tor was
transformed
contain
from p C a 2 A F
promoter,
a
plasmid
finally
contains
the
gene
C aM V
pla nt
neomycin
AFP
coding
c l one d
vector,
into
a
pMON200
a /3-glucuronidase
35S
construct
containing
m a tur e
cointeg ra tin g
P l a smi d pBI121
terminator.
in
fragment
35S
pBI1 21
11).
gene
levels
Hindlll
and NOS
vector
(Figure
(GUS)
wh ic h has
cloned
(Figure 10) .
The
the
resulting pIBI7 6AF was
pro m o t e r
expresses
cells.
Both
and
NOS
at
high
pBH21
and
phosphotransferase
(NPT
II)
resistance to the antibiotic kanamycin,
is useful for selection of the transformed plants.
To
determine
pMON200AF,
the
orientation
of
the
pBI121AF
and
pBI121AF and pMON200AF were digested with EcoRV,
and with Sstl and EcoRI,
respectively.
The digestion of plasm id pBI121 with EcoRV produces
fragments,
4.5-,
2.6-,
2.5-,
1.8-,
1.7-,
0.7-,
7
and 0.25-kb.
The 1.1-kb AFP gene cassette is inserted into the site of
1.7-kb
site
region,
between
sequence.
and
the
the
pBI 121AF
CaMV
35S
Therefore,
if
clone
promoter
contains
an d
the insertion
the
has
one
AFP
EcoRV
coding
a correct
A) pBI 121AF
35S
35S
Nos3*
Hind III
RB
<
AFP
LB
N P TII
NOS 5'
B) pMON 200AF
___
NOS 2 -
35S
GUS
NOS 3.
<
Nos3'
AFP
35S
35S
I
Hind III
N os-N ptll
N os
p o m o lo g y
^VUH
322
origin
/str
N o p au n e s y n th a s e
p tiT -3 7
Figure 11. Maps of the final clones, pBI 121AF
200AF (B).
(A) and pMON
48
or ie n t a t i o n ,
1.5-kb
a nd
(Figure
the
1.3-kb
12).
direction,
EcoRV
digestion
f ragments
If
the
the EcoRV
of
i n s t ea d
1 . 1-kb
pBI121AF
of
produces
1.7-kb
inse rt ion
has
of
an
pBI121
op posite
digestion generates 1 . 6-kb and 1.2-kb
fragments in the site of 1.7-kb of pBI121.
The digestion of p MON200AF clones with Sstl and EcoRI
g e ne r a t e
different
orientations.
d i g est ion
If
the
pat t e r n s
a cc o r d i n g
orientation
is
their
cor rect,
digestion produces 820-bp and 1090-bp fragments,
it should be 290-bp and 1620-bp fragments
to
the
otherwise,
(Figure 13).
2. Plant transformation and regeneration
1)
Agrobact er ium transformation
The A g r o b a c t e r i u m
used
for
the
transformation
re spectively.
conjugation
strains
Mating
system,
using
developed by Rogers et al
Agrobacterium
28°C for two days.
m e d i u m at 28 ° C .
was
of
LBA4404
pBI121AF
followed
the
and
by
helper
GV3111SE
and
the
were
pMON200AF,
triparental
plasmid
pRK2013
(1988).
LBA4 404
was
grown
in A B - B i o
medium
at
A g r o b a ct er iu m GV3111SE was grown in LB
AB-Bio m ed iu m was p re pared as follow:
49
<
0.5 k b ^ - 0.6 kb
35S
35S
AFP
Nos3'
Hind II! digestion
1
-<1 kb
— i ►« 0.7 kb ^
NPTII
35S
GUS
—
pBI 121
Hind 111 digestion
< - 0 . 6 kb — ^ 0 .5 kb
II
Nos3'
AFP
35S
>
35S
Figure 12. Determination of pBI121AF orientation. The two
possible orientations are shown here. Orientation I is the
proper type to be expressed in the plant system. The arrows
indicate the EcoRV restriction site.
Sst I
EcoRI
821 ----- J.290 >
I35S
35S
AFP
Nos3'
Hind III digestion
Sst I
800 b p ------3'
NPTII
LIH
5'
pMON200
Hind III digestion
II
AFP
Nos3
<290
t-
EcoRI
35S
- 821
35S
►t
Sst
Figure 13. Determination of pMON200AF orientation. Orientation
II is the correct type for expression in the plant.
51
AB-Bio medium;
10X AB salt;
The E . c o l i
and the E.coli
plus
and
to
glucose
lml
CaCl2.2H20
0.3g
MgS04.7H20
0.1ml
FeS04.7H20
100 ml
10X AB salt
lml
biotin
lOOmg
kanamycin per
30.0 g
K 2H P O 4
10.0 g
NaH2P04
10.0 g
NH4C1
1.5 g
KC1
c o n t ain ing
either
overnight.
log phase.
2 ml
of
sterile
10 m M M g S C M .
filter discs
The
mg/ml)
(25 mg/ml)
(0.2 mg/ml)
liter
pB I1 21A F
or
pMON200AF,
were grown
in LB medium
The cells were
diluted back
Then
cultures were mixed together,
(13
per liter
containing pRK2013
antibiotics
grown
5g
1 ml
fro m ea c h
of
three
spun down and resuspended in
mixture
on a fresh,
was
transfered
onto
n o n-d rie d LB agar plate.
The plate was incubated at 28 °C overnight.
The filters
were removed and placed into sterile tubes containing 2 ml
of 10 m M Mg S04 .
The solution was
vortexed to remove the
52
cells from the filter discs.
100 ul of the cells were then
p la ted onto an LB selection plate.
Transconjugants
were
selected on an LB plate containing 50 ug/ml kanamycin,
streptomycin for LBA4404
chloramphenicol,
and
containing pBI121AF,
50ug/ml
spectinomycin
for
kanamycin,
GV3111SE
and
and 25ug/ml
lOOug/ml
streptomycin
containing
pMON200AF,
respectively.
A gr ob ac te ri um transformation was further confirmed by
a
Southern blot of the
isolated A g r o b a c t e r i u m D N A .
Agrobacterium
plasmid
al
conjugated
(1988) .The
was
The
isolated by the m et hod of An et
Agrobacterium
was
grown
in
AB-Bio or LB mediu m containing an appropriate concentration
of antibiotics at 28 ° C .
lml of the overnight culture was
pell et ed in an Eppendorf
cells
were
r e s u s pen de d
(50mM
glucose,
lOmM
lysozyme) .
ad de d
and
in
incubated
0.1
EDTA,
After
temperature, 0.2ml of
centrifuge
25mM
10
of
10
II
i ce-cold
Tris-HCl
minutes
solution
for
ml
for 30 seconds.
solution
pH8.0,
at
room
0.2N NaOH)
room
1
4mg/ml
i n c u b a t i o n at
(1% SDS,
m i n ut es
The
was
temperature.
30ul phenol and 150ul of 3M sodium acetate p H 4 .8 were added
and
centrifuged.
ice-cold
95%
centrifugation,
The
ethanol
supernatant
and
was
precipitated
centrifuged.
with
After
the D NA pellet was washed with ice-cold 70%
53
ethanol
and
Tris-HCl,
resuspended
in
50ul
of
TE
buffer
(20
mM
1 mM EDTA).
2) Tobacco leaf disc transformation
The basic tobacco leaf disc transformation system was
followed
cells
(Horsch et al.,
(Nicotiana
tobacco
leaf
1988).
t a b a c c u m var
disc
Nurse cultures of tobacco
Xanthii)
were
transformation
to
us e d
in the
improve
the
transformation efficiency.
Tobacco cell suspension cultures were maintained in 50
ml
of
suspension
30 g/ l
sucrose,
culture m e d i u m
5ml/l
dichlorophenoxyacetic
(4.3g/l MS
B5
vitamins
acid
(2,4D)
stock
pH5.8)
salts
and
with
(Gibco),
lmg/1
a
2,4
constant
agitation of 150 rpm at room temperature.
B5 Vitamins stock solution;
The
lOOmg
myo-inositol
lOmg
thiamine-HCl
lmg
nicotinic acid
lmg
pyridoxine-HCl
two-week
old
tobacco
suspension
filtered through a 540um sieve mesh
clumps.
The filtrate
was
per ml
filter to
culture
remove
was
cell
then centrifuged at 4,000 rpm
54
for 20 minutes.
The cells were resuspended in M SO medium
(4.3 g/1 MS salts,
pH5.7).
The
and the
cells
in t e r f e r e s
r esu l t i n g
suspension
r es usp e n d e d
wi t h
the
regeneration
from
could
be
then
1 ml/1 B5 vitamins stock,
again
in M SO to wash
hormone
balance
explants.
used
was
The
for
30 g/1 sucrose
c ent ri fug ed
out
2,4
required
final
subculturing
D which
for
shoot
cell
su spension
and
leaf
disc
transformation.
To prepare nurse culture plates,
lml of the final cell
suspension was dispensed on each MS 104 m e d i u m plates
wi t h
7
g/1
phytagar,
1
mg / m l
b enc yl a d e n i n e ( B A )
(MSO
an d
0.1
mg/ml napthalene acetic a c i d ( N A A ) ) and covered with a piece
of Watman 3mm filter paper.
Leaf
discs
(1cm diameter)
plants asceptically grown
wer e
punched
discs.
The
with
leaf
a
ob ta i n e d
in magenta boxes.
sterile
discs
were
were
cork
borer
precultured
from tobacco
Young leaves
to
produce
for
1 or
leaf
2 days
upside down on MS104 m ed ium plates to allow initial growth.
10
ml
of
an
c e n t r ifu ge d
overnight
for
culture
10 minutes
at
of
4,500
A .t u m e f a c i e n s
rpm.
The
were
ba cterial
pellets were resuspended in MSO to a final concentration of
1 0 8/ml.
resuspended
Explants
cult ur e
were
of
soaked
for
few
seconds
A .t u m e f a c i e n s , b l o t t e d
pl aced upside down on MS104 nurse culture plates.
dry,
in
a
and
55
After
3
tr a n s f e r r e d
d ays
to MS
carbenicillin
or
of
co-culture,
selection m e d i u m
cefatoxime,
visible,
the
rooting
medium
were
500
ug/ml
kanamycin)
for
When defined stems were
(MSO
with
0.6%
phytagar,
100 ug/ml k a n a m y c i n ) .
roots
then
ug/ml
wit h
were
shoots were excised and p l a c e d upright
carbenicillin,
developed
explants
(MS104
300
selection of tr ansformed callus.
the
after
about
transferred
to
two
ug/ml
The plantlets which
weeks
Magenta
500
in MS
in
rooting
boxes
m e diu m
containing
MS
rooting medium.
When the plants developed extensive roots, the lid was
opened
for
3
environment.
days
to
acclimate
The plants
the
plants
were then t r a n s f e r r e d
pots containing P r o -M ix soil,
to
to
the
Jiffy
and then to 10 inch pots and
grown in a greenhouse under standard conditions.
3)
Determination of the segregation ratio of kanamycin
resistance gene
Transgenic tobacco plants grown in the greenhouse were
wa t e r e d
ever y
other
day
Gro w fertilizer biweekly.
early
floral
stage,
with brown paper bags
the
and
supplemented
wi t h
When the plants
tobacco
flower
buds
a
Miracle
rea ch ed at an
were
to insure self-pollination.
covered
After
5B
six weeks,
ripe capsules were removed and further dried at
37 °C.
Seeds
inc uba ti on
0.1%
20
wi t h
sucrose,
in MS
gentle
cheese cl oth
in
10%
and
agitation.
plate
steri li zed by
com mer cia l
(4.3
bleach
with
seeds
were
The
with sterile water.
m inimal
0.8% agar)
for
in
m i n u tes
several times
sown
select
wrapped
for
Tween-20
washe d
then
were
The
g/1
MS
seeds were
salts,
10
g/1
supplemented with 100 mg/1 kanamycin to
kanamycin
gene
resistance.
The
seeds
were
3000
lux.
grown in environmental growth chambers at 25 °C,
Seeds which did not carry the kanamycin resistance gene had
white leaves and died after four to eight weeks.
3.
P-glucuronidase
The
presence
t r a n sg en ic
plants
described
by
diameter)
GUS
of
was
P-glucuronidase
assayed
Jefferson
by
the
(1987).
activity
flu ro me tr ic
Leaf tissue
punched with a cork borer was ground in 500
extraction
buffer
p-mercaptoethanol,
sarcosine,
assay.
(GUS)
0.1%
lOmM
T r ito n
(50mM
Na2
the
me th od
(15mm
ul of
NaPO-3 p H 7 . 0 ,
EDTA,
X-100) .
in
50
lOmM
0.1% s o d i u m lauryl
ul
of
extract
was
incubated at 37 °C in 500 ul of assay buffer containing ImM
57
4-methyl
umbelliferyl
intervals,
|3-D-glucuronide
(0.2M Na2C03) .
4-methylumbelliferone
nm,
and
(MU)
regular
of
at
(Hoefer
protein concentration
Bradford
The fluroscence of liberated
was me asured with excitation at
emission
sp e c t r o f l u r o m e t e r
method
At
100 ul of the reaction mixture was added to 0.9
ml stop buffer
365
(MUG) .
455
nm
using
S c i ent if ic
Inst.,
a
TKO
100
CA) .
Total
in leaf tissue was determined by the
(197 6)
us i n g
the
P i erc e
BCA
p r o te in
assay reagent.
4.
DNA analyses
1)
Total plant DNA isolation.
To check
for stable
tobacco plant
genome,
the pr ocedure
of Chen
ground
fine
with
The
propylene
e x t r act ion
Tris-HCl
sarkosine,
b u ffe r
p H 8 .0,
(1986).
190
ml
added up to 12 ml.
p owd er
tube
(168
16
of AFP
gene
tobacco genomic DNA was
liquid nitrogen
powder.
Falcon
integration
ml
in a mortar and
was
water).
transfered
contained
urea,
0.5
isolated by
Two g fresh tissue
which
g
into the
25
M
ml
EDTA
5M
pestle
into
6
ml
pH8.0,
shaken
to a
15
of
NaCl,
Phenol/chloroform
The tube was
a
20
20
was
ml
ure a
ml
1M
ml
20%
(1:1)
was
hard to mix the
58
two
p has es
well
to
a
thick
p as te
and
in cu b a t e d
at
room
temperature for 15 minutes.
The mixture was centrifuged at 8K for 10 minutes at 4
°C.
The supernatant was filtered thro ug h a small piece of
mi r a c l o t h to get
ammonium
rid of floating particles.
acetate,
pH5.2,
and
isopropanol
added to precipitate the DNA.
table top
The
centrifuge
pellet
was
One ml
to
13
ml
4 . 4M
were
The D NA was spun down in a
at the highest
transferred
into
dissolved with 500 ul of TE.
speed
an
for 3 m i n u t e s .
Eppendorf
tube
and
The DNA was precipitated by
adding 100 ul 4.4M ammonium acetate and 0.7 ml isopropanol.
The D NA pellet was collected by microfuge for 5 minutes and
washed
with
75%
ethanol.
The
DNA
pellet
was
dried
and
dissolved in 200 ul TE buffer.
2)
Preparation of hybridization probes.
Hybridiza ti on probes
pr i m e d
DNA
labelling
Laboratories
1111-bp
kit
(IN) .
Hindlll
The
fragment
were pre p a r e d by using
a random
supplied by
Ma nn h e i m
174-bp
Bo e h r i n g e r
mature
of p BI1 2 l A F
AFP
were
DNA
u se d
and
the
for probe
preparation.
The D NA fragment
10 minutes
at
denatured D NA
95 °C
(25ug)
and
was mixed
was
dena tu red by heating
subsequent
with 3 ul
cooling
on
for
ice.
The
of dNTP mixture
(dATP,
59
dGTP,
d T T P ) , 2 ul
dCTP
to
of
reaction buffer,
(3000 C i / m m o l e ) .
19
ul
with
added.
min ute s
and
re ac tio n
and
rea c t i o n
stopped
5ul
of
[a-32P]
The reaction mixture was brought up
wa te r
The
and
was
by
mixture.
then
1 ul
c a r ri ed
a ddi ng
The
of
2
prepac ke d Sephadex G-50 column
out
ul
labeled
Klenow
of
DNA
at
37
0.2M
was
enzyme
°C
for
EDTA
to
se pa r a t e d
(5 prime-3 prime,
was
30
the
on
INC.,
a
PN)
as described in the manufacturer's manual.
3)
Southern blot
Southern
i n corporation
tobacco
analyses
of the
plant
Hindlll.
were p e rfo rm ed to
AFP
genomes.
gene
The
check the
into
the A .tu me fa ci ens
DNA
samples
For A g r o b a ct er iu m DNA,
were
cut
Hindlll
were
used
every
of
DNA.
with
with
digestion,
the DNA was run on 1% agarose gel.
RNase
was
Hindlll
After staining the gel with ethidium bromide,
the gel
in
1. 5M
NaCl,
neutralized in 1.5M NaCl,
Transfer of DNA to a
reaction.
10 units
After
denatured
digestion
2 ug
incubated
was
the
for
a nd
10 units of Hindlll were
used for every 4 ug D N A and for plant genomic DNA,
of
stable
0. 5M
Na O H
0.5M Tris-Cl,
for
pH 7.0,
1
hour,
and
for 1 hour.
nitrocellulose filter was done by the
traditional capillary blotting procedure using the 10X SSC
BO
(87.5 g/1 NaCl,
44.1
by Maniatis et al
g/1 Sodium citrate pH
(1982).
After transfer,
7.0)
described
the filter was
washe d with 6X SSC for 5 minutes at room temperature,
dr ie d on a sheet
of
3MM paper,
and b a k e d under
air
v a c u u m at
80°C for 2 hours.
The
hours at
SDS,
nitrocellulose
68 °C
0.2ml/cm2
was
in prehybr idi za tio n
5X Denhardt's
sperm DNA) .
f il te r
(6X SSC,
of pre hy bridization
nitrocellulose
prehybridization
solution
for
2
0.5%
solution and 100 ug/ml d enatured salmon
The amount
of
prehybridized
so lution
incubated
with
32P - l a b e l e d
denatured
was
filter.
poured
hybridization
prob e
solution was
After
off,
the
solution
D NA
in
the
filter
(lOmM
was
EDTA,
p r e h y b r idi zat io n)
at
68°C for 12-16 hours.
After
h y b r i d iz at ion ,
the
filter
solution of 2X SSC and
0.5%
minutes,
a solution
for
15
filter
and then
minu tes
was
with
with
incubated
was
washed
SDS at room temperature
occassi ona l
of
2X SSC
gentle
in a solution
of
dr ied
paper,
at
room t e m p e r atu re
wrapped in Saran Wrap,
on
a
and
0.1X
SSC
sheet
of
a
for 5
0.1%
agitation.
SDS at 68°C for 2 hours with gentle agitation.
was
with
and
SDS
The
0.5%
The filter
Whatman
3MM
and applied to X-ray film to
obtain an autoradiographic image.
61
5.
RNA
analyses
1)
Total tobacco RNA isolation.
To
determine
transgenic
tobacco
t he
tobacco
plants,
leaf tissues
(1987).
total
of
RNA
the
was
AFP
gene
isolated
in
from
using the protocol of Logemann et al
Leaf tissues
and ground,
expression
(2g)
were frozen in liquid nitrogen
using a mortar and pestle.
The tissue powder
was h o m o g e niz ed by the addition of 2 volumes
of guanidine
buffer
4-m orpholine
(8M
g u an id ine
ethansulfonic acid
pH 7.0) .
The
centrifuged
in
hydrochloride,
(MES), 20mM EDTA,
guanidine
a
20
mM
50mM P-mercaptoethanol
hydrochloride
precooled
c ent rifuge
for
extract
10
minutes
was
at
10.000 r p m .
The
volume
supernatant
of
proteins.
was
mixed
vigorously
w it h
phenol/chloroform/isoamylalcohol
The mixt ure
was
c e n t r ifu ge d
for
to
0.2-1.0
r em ove
45 minutes
10.000 rpm at room temperature to separate the phase.
at
The
RNA-containing aqueous phase was collected and precipitated
with p r eco ol ed 0.7 volume of ethanol and 0.2 volume of 1 M
acetate
at
-20°C
overnight.
pell e t e d at
10,000 rpm for
ethanol.
The
water.
R NA
was
The
precipitated
10 minutes
drie d
and
RNA
was
and washed with
70%
dissolved
in
sterile
2)
Northern blot.
The formaldehyde R NA gel electrophoresis procedure as
described
pr e p a r e
by
Maniatis
the
(3.0%)
was
et
al
fo rma l d e h y d e
cooled
to
(1982)
gel,
a nd
acid
acetate,
and
5mM E D T A
melting
60°C,
morpholinopropanesulfonic
pH8.0)
was
5X
(MOPS)
2ul of
5X gel-running buffer,
formamide,
agarose
gel
in
(0.2M
50mM
sodium
were
a dd ed
bromophenol blue,
to
was m ix ed with
formaldehyde
and lOul
and incubated at 55°C for 15 minutes.
ul of sterile loading buffer
water
respectively.
(up to 20 ug)
3.5ul
To
buffer
pH7.0,
f o rma ld ehy de
give IX and 2.2M final concentrations,
4.5ul of the R N A sample
followed.
(50% glycerol,
0.4% Xylene cyanol)
Then,
ImM EDTA,
2
0.4%
was added to the RNA
sample mixture.
The RNA samples were loaded onto the gel.
electrophoresis,
membrane
manual.
the
RNA
(Stratagene,
The
gel
minutes each time.
was
of
tran sfe red by
CA)
was
t r a n s fe rre d
was
Dura lo n-U V
according to the manufacturer's
r i nse d
twice
0.05M NaOH,
wit h
water,
for
5
0.15M NaCl,
the
and then for 30 minutes
0. 1M Tris-HCl,
traditional
the 10X SSC as the transfer buffer.
membrane
onto
The gel was soaked for 30 minutes in a
solution of 0.15M NaCl,
in a solution
was
After gel
blotted
of
vacuum for 2 hours at 80°C.
excess
pH8.0.
RNA
capi ll ary m e t h o d
using
After transfer,
buffer
a nd
baked
the
under
63
The
membrane
wa s
prehybridized
containing 50% deionized formamide,
in
a
solution
10% dextran sulfate,
1%
S D S , 1M NaCl and 100 ug/ml denatured sonicated salmon sperm
D N A for
1 hour at
42°C with
constant
agitation
in a heat
sealable bag.
After prehybridization,
a n d the bag
was
the probe was added in the bag
incubated overnight
specific activity was
1 to
at
42°C.
5 x 105 c p m / m l .
The probe
The membrane
was washed one time at room temperature for 15 minutes with
a solution of
and
the
2X SSC,
0.1% SDS to remove any unbound probe
hybridization
solution.
washed with a solution of 0.IX SSC,
minutes.
At this time,
The
membrane
was
0.1% SDS at 65°C for 15
the membrane was monit or ed with a
Geiger counter to decide about further washing steps.
membrane
then
was blot ted off with
filter paper
The
and exposed to
X-ray film.
6.
Protein analyses
1)
Silver staining of total tobacco protein.
The total protein of tobacco plants was extracted from
leaf tissues
with extraction buffer
5mM DTT,
0.05%
Triton
Tobacco
leaf tissues
X-100,
were
(50mM Tris-HCl pH7.5,
50mM EDTA,
ground
0.19 mg/ml P M S F ) .
in liquid nitrogen and
64
centrifuged
at
supernatant
10,000
was
r pm
for
collected
30
m i n ut es
at
4°C.
and
the
total
The
protein
concentration was determined using the Biorad protein assay
reagent.
minutes
Tris,
5
ug
of
protein
in a loading
2%
solution
mercaptoethanol,
sample
were
(4% SDS,
0.1%
boiled
for
12% glycerol,
b rom op h e n o l
blue
5
50mM
pH6.8).
The Mini-PROTEAN II Dual Slab Cell from Bio-Rad was used to
run
the
16.5%T,
separate
t he
3%C
Tricine-SDS
proteins
(Herman
polyacrylamide
et
a l .,
gel
1987) .
to
The
electrophoresis was run at 150 V for 60 minutes.
The
performed
s ilver
w it h
staining
the
electrophoresis,
solution
of
Bio-rad
the gel was
(40% methanol,
the
p o l y a c r y l a m i d e gel
s il ver
stain
soaked
kit.
was
Aft er
in 200 ml of fixative
10% acetic acid)
for 30 minutes.
The gel was further fixed two times in 200 ml of a solution
containing 10% ethanol/5% acetic acid for 15 minutes
After
fixing the gel,
followed
yellow
by
color
stained with
minutes.
washing
was
the
wi t h
100 ml
gel was oxidized for 5 minutes,
deionized
removed
from
the
silver reagent
After washing
each.
wat er
gel.
unti l all
the
The gel
was
(1:10 dilution)
for 20
the gel with deionized water,
the
gel was deve lo ped with developing solution until the bands
appeared
dark
brown.
Development
was
stopped
incubating in 5% acetic acid solution for 5 minutes.
by
65
2)
Western blot
After electrophoresis as described above,
we r e
transferred
Hoefer's
to
Semip hor
Immobilon
S emi - D r y
PVDF
Transfer
the proteins
membrane
Unit.
using
a
The m em brane
and Whatman 3MM filter paper were equilibrated in blotting
buffer
10
(192 m M glycine,
m inutes,
t r a nsp ho r
after
sandwich
instructions.
25mM Tris pH 8.3,
wetting
was
The
in
100
co n s t r u c t e d
H oef er
%
20% methanol)
methanol.
according
semiphor
unit
to
was
constant current of 100 mAmp for 30 minutes.
was
air
dried
a nd
stored
at
4 °C
for
until
The
Hoefer's
run
at
a
The membrane
ready
to
be
immunostained.
After
re-wetting
membrane was brie fly
solvent
and
co nt aining
7.4)
for
then
5% B S A
1
hou r
in
methanol
rinsed
in
in
(0.9% NaCl
at
37 ° C
a
protein
with
membrane was w ashed three times
wash
solution
incubated
(0.1% B S A
wi t h
the
1-2
seconds,
in water to remove the
placed
TBS
for
excess
s olution
in 20 m M Tris-HCl
g en tle
agitation.
for 5 minutes
in TBS) .
purified
blocking
the
anti-IgG
The
each
The m emb ra ne
raised
pH
in the
was
then
again st
a
winter flounder antifreeze peptide in a volume of 10 ml of
antibody incubation buffer
for
2 hours
The membrane
at
room
was
(1% BSA,
tempe ra tur e
washed
as
0.05% Tween-20
with
described
gentle
in TBS)
agitation.
above,
and
then
66
i n cub ate d
with
phosphatase
the
(AP)
roo m temperature
secondary
conjugate
antibody,
a nti -I gG- Al kal ine
(1:7500 dilution),
in 10 ml of antibody
for 1 hour at
incubation buffer.
The membrane was washed again as described above.
The
AP
color
5-bromo-4-chloro-3substrate.
mixing
reaction
was
indolyl-phosphate
AP color development
66 ul of
33 ul of BCIP
100 m M NaCl,
in
10 ml AP buffer
the AP color development
The
(BCIP)
using
as
the
solution was prepa red by
nitro blue tetr az oli m
5mM MgC12) .
t u r n e d purple.
performed
(NBT 50 mg/ml)
(lOOmM Tris-HCl pH
The m embrane
was
and
9.5,
incubated
in
solution until the reactive areas
reaction was
stopped by washi ng
the
membrane with distilled water.
3)
Purification of IgG
The winter
provided
by
Bi o p h y s i c s ,
Dr.
flounder antifreeze-protein antiserum was
D e V r ies
University
w as
(Department
of
of
Illinois).
-protein
(IgG-AF)
purified
immunopure
IgG purification kit.
by
Physiology
and
IgG-antifreeze
using
t he
Pierce
A Protein A A ffinity Pak
column was equilibrated with 5 ml of Immunopure IgG binding
buffer.
Antiserum
was
dilut ed
1:1
with
Immunopure
binding buffer and then applied to the column.
IgG
The column
67
was
washed
with
15
ml
of
Immunopure
binding
buffer,
followed by eluting the bound protein with 5 ml of elution
buffer.
The 5 ml eluent was precipitated with an equal volume
of saturated am monium sulfate
solution at 4 C overnight.
The precipitate was c entrifuged at 3,000
and
the
p e ll et
phosphate,
was
resuspended
lOOmM NaCl pH7.4).
in
1ml
g for 30 minutes
PBS
(20mM
The p rec ip i t a t e d
so dium
antibody
solution was desalted in an Excellulose column equilibrated
with 10 ml PBS.
was
A 1 ml sample of the precipitated IgG-AF
appl ied to the
were collected.
Excel lul os e
The
column
and
1 ml
fractions
IgG-AF concentration was m o n it ore d by
absorbance at 2 80 nm.
7. Frost test.
The measurement
of
frost tolerance
of tobacco plants
was followed by the procedure of Lindow et al
little modification.
the 4 leaf stage)
containing
a
Kanamycin selected seedlings
jiffy
tablet.
growth
to p
lid.
the
synchronously,
so
with a
(about
were transferred into Magenta boxes,
en vironmental
of
(1982)
The plants
chamber until
Tobacco
the
were
the plants
seeds
developmental
do
not
stage
grown
each
in an
reached the
germinate
of
all
the
68
s e e dli ng s
placed
was
not
randomly
incubator.
temperature
precisely
at
After
was
0°C
30
lowered
in
the
the
m i n ute s
same.
plants
temperature
equilibrium
rapidly
to
-4°C,
cooled to -7°C at a rate of -l°C/20 min,
30°C and kept overnight to recover.
were counted visually.
The
were
controlled
at
0°C,
then
the
slowly
then increased to
The surviving plants
69
Results and Discussion
1. Construction of recombinant DNA
The IIA7 cDNA clone contains the whole winter flounder
antifreeze-protein
(AFP)
mature
regions
poly
signal
sequences
and
gene
sequence:
d(G)
g ov e r n e d
and
by
fish
poly
pre-,
d(C)
hormones
necessary for expression in the plant
pro-,
and
tails.
would
system.
The
not
be
Therefore,
the 174-bp mature AFP gene was designed to contain its own
initiation and termination codons and inserted into plasmid
pBR322 digested with BamHl.
of
a
correctly
re sultant
pBR322AF
size d
plasmid
was
Figure 14 shows the presence
band
p BR3 2 2 A F
amplified
in
of
174-bp
derived
after
BamHl
large
q u a nti tie s
fr o m
digestion.
for
the
The
further
cloning into suitable plant transformation v e c t o r s .
The
cloned
174-bp
into
plasmid
mi n i s c r e e n e d by
clones
15).
mature
12
(CaMV)35S/AFP/NOS
gene
fro m
pM ON530.
digestion
showed bands
Clone
AFP
with
Twelve
Ec oRV
of the correct
showed
3'
gene
resulting plasmid was named
the
the
colonies
and EcoRl,
size of
correct
cassette
pMON530AF.
pBR322AF
324-bp
and
were
four
(Figure
orientation
(Figure
was
16) .
of
The
Figure
14 .
174-bp BamHl
1-4: pBR322AF
Agarose
fragment
gel
of AFP
showing
gene
digested with BamHl,
lane 6: 123-bp ladder.
the
presence
from pBR322AF.
lane
5: pBR322
of
Lanes
alone,
71
Figure
324-bp
Agarose
EcoRV-EcoRl
1,2,4,5:
lane 3:
15.
pMON530AF
gel
fragment
clones
123-bp ladder.
showing
the
of pMON5 30A F
presence
clones.
of
Lanes
digested with EcoRV and EcoRl,
72
1 2 3 A 5 6
- -
Fi gure
16.
pMON530AF.
pMON530AF
The
Agarose
The
clones
di ge st io n
-
’S f i l
gel
324-bp
were
with
showing
the
EcoRV-EcoRl
isolated
SfaNl
lane
clone
2:
generates
6,
134-bp
lane
123-bp ladder.
5 without
4:
clone
digestion
7,
lane
and
of
from
SfaNl.
190-bp
and 280-bp and 44-bp
for an incorrect orientation.
clone
fragments
and digested with
fragments for a correct orientation,
fragments
orient at ion
with
5:
Lane
1:
SfaNl,
clone
12,
clone5,
lane
3:
lane
6:
73
The
fi na l
clones,
pBI121AF
pMON200AF
(c o i n t e g r a t i n g
restriction
enzyme
Hindlll
d ig es tio n
the AFP
gene cassette;
promoter,
were
confirmed
and Southern blotting.
pB I12 1A F
excised an approxim at ely
vector)
vec to r ),
digestion
of
(binary
and
p M O N2 00 AF
l.lkb band which
that
is,
the
(Figure
and
by
The
17)
corresponded to
duplicated
the mature AFP gene coding sequence,
(CaMV)
35S
and the NOS
3' t e r m i n a t o r .
The correct orientation of pBI121AF and pMON200AF was
dete rm ine d
accord ing
to
whic h
described
in
are
pBI121AF,
(lanes
(lanes
the
2,3,5),
1,4)
Agrobacterium
clones
clones
or
else
(Figure
12
M a te r ia ls
ei th er
usin g
showe d
the
18) .
13,
an d
Methods.
labe led
respectively,
For
correct
o r i e n t at io n
opp o si te
or ie nt at io n
Clone
orientation
the
and
s how ed
transformation.
showed correct
hybridization
figures
For
6
was
used
pMON200AF,
(Figure
174-bp
19).
AFP
all
for
the
Southern
DNA
further
confirmed the presence of the AFP gene in the final clones,
p B I 121AF and pMON200AF
(Figure 20).
2. Transformation of Agrobacterium tumefaciens
Using the triparental
mating procedure,
pBI121AF
pMON200AF were transformed into the disarmed A.
and
tumefaciens
74
Fi g u r e
17.
1 . 1-kb Hindlll
plasmids
agarose
pBI121AF,
were
gel.
Agarose
fragment
gel
s ho win g
the
presence
of
of pBI121AF
and pMON200AF.
The
digested with Hindlll
and run on the
0.9%
A)
lane
1:
1-kb
lane 7: pBI121 only.
lane 5: pMON200 only,
size
B)
marker,
lanes
1-4:
lane 6: 1-kb size marker.
lanes
2-6:
pMON200AF,
75
1 2
F ig ure
pBI121AF
The
and
1:
Agar ose
clones.
EcoRV
1.3-kb
18.
di ges tio n
clone
clone
pBI121AF clone
marker.
of
for
1.2-kb fragments
pBI 121AF
gel
showing
The plasmids were
fragments
pBI121AF
3 4 5 6 7
5,
1,
a
p B I1 2 1A F
correct
the
o ri en tat io n
digested with EcoRV.
g e n er at es
1 . 5-kb
orientation,
and
lane
lane
lane
4:
2:
pBI121AF
pBI121AF
6: pBI121 only,
clone
clone
6,
3,
lane 7:
and
1 . 6-kb
for an incorrect orientation.
7,
of
Lane
lane
3:
lane
5:
1-kb size
Figure
pMON200AF
19.
Agaro se
clones.
gel
showing
The plasmids
were
the
or ie nt at ion
digested with
and EcoRl and run on the 0.9% agarose gel.
p ro d u c e s
1620-bp
orientation,
incorrect
and
29 0-b p
1090-bp
and
orientation.
approximately
EcoRl.
and
Lanes
800-bp
1-4:
6 : 1-kb size marker.
ban d
fr agm ent s
820-bp
for
a
correct
fragments
pMON200
with
d ig es tio n
lane
Sstl
The digestion
The
pMON200AF,
of
for
produces
of
5: pMON200
Sstl
only,
an
an
and
lane
77
t
Figure
plasmid
Hindlll
Lane
20.
DNAs.
Southern
The
1: pBI121,
4: pM0N2 0 0 A F .
hybridization
plasmid
and hybridized
with
'
DNAs
the
were
labeled
lane 2: pBI121AF,
analysis
d i ge st ed
of
with
174-bp AFP
DNA.
lane 3: pMON200,
lane
78
strains LBA4404 and GV3111SE,
resistant
to
kanamycin
LBA4404-containing
kanamycin,
respectively.
and
pBI121AF,
GV3111SE-containing
streptomycin
and
streptomycin,
to
and
pMON200AF,
for
chloramphenicol,
spectinomycin
were
Agrobacterium DNA was isolated.
The colonies
se l e c t e d
for
and
total
Stable transformation was
confirmed by Southern hybridization with the labeled 17 4-bp
AFP
DNA probe.
22.
Hindlll digestion of total Agr oba ct eri um DN A excised a
1 .1-kb
band
The
results
corresponding
are
to
shown
th e
in
figures
sequence
21
and
of
the
double-(CaMV)35S/AFP/NOS 3' gene construct.
3.
Transformation
of
to b a c c o
p la n ts
with
tumefaciens
After infection of leaf discs with LBA4404-containing
p B 1 1 2 1 AF
pMON200AF
(L B A 4 4 0 4 / p B I 1 2 1A F )
and
(GV3111SE/pMON200AF), calli
most of the
leaf discs.
The
GV3111SE-containing
started to form from
leaf discs
were transferred
to shoot-inducing media containing kanamycin,
eight
shoots
and 24).
per
disc
were
These kanamycin
to become healthy plants
thereby
devel ope d
and three to
(Figures
23
selected shoots were regenerated
-(Figure 25) .
Kanamycin resistance
A.
79
Fi gu re
21.
A .t u m e f a c i e n s
integration
negative
with
the
LBA4404
positive control
DN A
DNA
control
tumefaciens
hybridization
LBA4404/pBI121AF.
of AFP
agrobacterium
hybridized
S o u th er n
of
transformed
To
confirm
the
into A. t u m e f a c i e n s
LBA4404,
the
Hindlll
a nd
wa s
digested
label ed
with
174-bp
(LBA4404/pBI121),
tr an sf or med
(pBI121AF).
with
AFP
DNA.
la nes
pBI121AF,
Lane
2-7:
lane
1:
A.
8:
Fi g ur e
22.
Sou t he rn
tumefaciens
GV3111SE
co n fir m
in tegration
the
GV3111SE,
hybridization
tr an sf or med
of
AFP
with
DNA
an al y si s
pMON200AF.
into
of
A.
To
A .t u m e f a c i e n s
the agrobacterium DNA was digested with Hindlll
and subjected to Southern hybridization analysis with the
174-bp
AFP
D NA probe.
2-5: GV3111SE/pMON200AF.
Lane
1:
GV3111SE/pMON200,
lanes
Figure 23. Leaf disc transformation.
discs
were
t r a n s fo r me d
grown
shoots were
in
with
A .t u m e f a c i e n s
s ho ot -i n d u c i n g
removed
to rooting medium.
from the
medium.
leaf discs
A)
Tobacco leaf
LBA4404/pBI121AF
B)
Individual
and transferred
82
F ig ur e
24.
A. t u m e f a c i e n s
T o ba cc o
leaf
disc
GV3111SE/pMON200AF.
leaf discs on shooting medium
on rooting selection me di um
transformation
Tra nsf or me d
with
tobacco
(A) , and transformed shoots
(B) .
Figure 25. Transgenic tobacco plants.
84
was the selection marker of the transformed plants,
because
the
contain
int roduced plasmids,
pB I12 1 AF
the neomycin phosphotransferase
and
pMON200AF,
(NPT II) gene which confers
resistance to the antibiotic kanamycin.
Re searchers
have
reported
that
a
t r a n sf or m ed
callus
does not necessarily regenerate transformed shoots
and McHughen,
plants
1988a).
developed
actually
from
transformed
screening,
based
An d indeed,
on
the
the kanamycin-selected
transformed
themselves.
kanamycin
from non- tra ns for med
cells
ca l li
Escapes
r es ist anc e,
occurred in the nontransformed plants.
arise
(Jordan
are
not
from
the
mi g h t
have
Escapes most likely
cr os s- pro tec te d
from the
selective agent by transformed cells in the callus.
Therefore,
as another marker for plant transformation,
P~glucuronidase
(GUS)
activity
was
measured
regenerated plants with LBA 440 4/ pB I1 21 AF .
plants
GUS
tr an sf or med
ac t i v i t y
even
with
all
the
plants
resistant.
Table 4 shows the expression
gene
t r a n sf or me d
in
va r ie d
the
in
act ivity
the
each
may be
chromosomal
of
the
plants.
plants.
att ributed
integration
gene copy numbers,
to
The
The
several
site
D NA methylation,
of
and
showed
negative
were
kanam yci n
level of the GUS
GUS
act ivi ty
dif fe re nc e
factors,
the
the
Eight out of 33
L BA 44 04/ pBI 12 1AF
t ho u gh
in
in
was
GUS
including
intr odu ce d
gene,
inherent variation
85
Table
plants.
4.
The
GUS
GUS
methylu mb ell if ero ne
leaf
disc
glucuronide
with
ac ti vit ie s
ac tiv ity
(MU)
an
was
of
measured
control
as
pr odu ce d per minute
excess
4-
nM Mu/ cm2 , min
ND
LI
70. 9
L3
388. 9
L5
76.0
ND
L13
291.1
L17
26.4
L24
ND
L25
39.3
L2 6
93.8
L2 9
35.3
L30
86.3
L32
389. 6
toba cc o
a nmole
in the
4-
1cm2
methylumbelliferyl
(MUG).
Plants
L12
t ra n s g e n i c
86
in leaf sampling.
The
gene
most
likely
e x p r es si on
integration
position
is
in the
gene
can
near
influ enc e
foreign
ge ne
sequence,
the
the
ne ar -b y
q u a n t it a ti ve
the exi st e nc e
The
of
d if fe ren t
effect
cis -elements
is
is
in
sites
known
well
of
as
the
known
to
in Agrrobacteri urn-mediated plant
The p o s i t i o n e ff ec t
expression
v a r ia ti on
a phenomenom
p os it i on
expression
transformation.
insertion
of
chromosome,
effect.
influence
cause
(positive
from
integrated
the
may
or
be
due
negative)
tra nsgene.
near
th e
to
which
If
plant
the
enhancer
introduced gene may be highly tr anscribed by
en h a n c i n g
trans-f act ors
and
factor.
cis-elements
The
of
the
i nte ra cti on
between
intr odu ce d
DN A may
also be influenced by the site of integration.
DN A
m e t h y la ti on
in
plants
correlated to gene expression.
varies
and
between
inactive
reg io ns.
the
GUS
sequence
be expected.
gene
shown
to
be
inversely
The degree of methylation
actively transcr ibe d
inactivate
coding
is
Highly
regions
of the
methylated
expression.
If the
are methylated,
low GUS
genome
DNA
regions
can
in the
activity will
87
4.
Integration
of
the
mature
AFP
gene
tobacco plants
Southern
blo t
analysis
unambiguously
integration of the mature AFP
gene
proves
into the plant
the
genome.
All transgenic plants transformed with LBA4404/pBI121AF had
a
complete
Hindlll
corresponding
to
fragment
th e
/mature AFP/NOS 3'
gene
a pp ro xi mat ely
construct,
(Figure 26).
L12 and L24 plants,
Hindlll
(lanes
5 and
gene
by
their
kanamy cin
1.1-kb Hindlll
may be
gene
8) .
This
rea rra nge me nt
resistance.
As
or
1.1-kb DNA probe.
double-(CaMV)35S
may
indicate
that
or contain only the
del etion
the
fragment of pBI121AF,
hyb rid iz ed with
size
did not excise the 1.1-kb
plants L12 and L24 are not transformed,
NPTII
in
Those which had negative
GUS activity,
band
1.1-kb
probe
used
the plant
the N 0 S 3 1 sequence
beca use
was
of
the
genomic DNA
of the
labeled
This would explain the presence of the
high molecular size band in the blots.
Ten
out
of
GV3111SE/pMON200AF
the
1.1-kb
control
13
transformed
with
showed a distinct band corresponding to
ant ifreeze
plants
plants
gene
tr a ns fo rm ed
excise the 1.1-kb band.
construct
with
(Figure
27) .
GV 3 11 1SE /pM ON 200
did
The
not
into
8B
1 2
3
4
LI
Figure
toba cc o
genome,
26.
174-bp
control
3-13:
Southern
9 tO
plants
with
DN A
analysis
of AFP
DNA
into the
of
To
tobacco
isolated from transgenic tobacco
probe.
transformed
tr a n s f o r m e d
L29 L30 132
LBA4404/pBI121AF.
digested with Hindlll
AFP
11 12 13
hybridization
integration
DNAs were
leaf tissues,
the
8
7
tr an sf or me d
the
plant
6
L5 L12 L13 L17 L24 L2S L26
plants
demonstrate
5
pl ants
and hyb ridized with
Lanes
with
with
1
a nd
2:
neg at iv e
LBA4404/pBI121,
lanes
LBA4404/pBI121AF.
The
arrow shows the distinct band corresponding to the 1.1 kb
Hindlll
fragment
(C a MV )3 5S / A F P / N O S 3 ' gene
containing
the
construct.
letters
The
double
on
bo ttom of the blot indicate the specific plants used.
the
G13G12G11 CIO G9 G8 G7 G6 G5 G4 G3 G2
Figure
27.
Southern
hybridization
analysis
of
tobacco plants transformed with G V 3 1 1 1 S E / p M O N 2 0 0 A F .
The
tobacco
and
genomic
DNAs
digested with Hindlll.
DNA.
La n es
1,2:
G V 311 1S E/p MON 20 0,
were
is o l a t e d
from
leaves
The probe used was the 174-bp AFP
control
lanes
plants
3-15:
shows
transformed
with
The
distinct
band
corresponding to the
1.1-kb antifreeze gene insert.
The
on the bo ttom of the blot
plants used.
the
wi t h
GV3111SE/pMON200AF.
letters
arrow
plants
transformed
indicate the
specific
90
The GUS assay and Southern blot analysis indicate that
the efficiency of plant transformation was almost the same
between
plants
vector
infected
system)
spurious
and
into
LBA4404/pBI121AF
GV3111SE/pMON200AF.
rearrangements
process
with
the
o c cu rr ed
plant
GV3111SE/pMON200AF
infected
numbers
gene
of the AFP
In addition,
during
the
than
that
However,
showed
of
no
int egr at io n
chromosome.
plants
(binary
the
hi g h e r
plants
copy
infected
with LBA4 4 04/pMON200AF.
As each of these transgenic plants
independent
may
transformation,
differ
in
integrated
in to
recombination,
expression
plants
by
each
plant.
the
plant
modifying
the
genome
site
of T-DNA
in tr odu ce d
by
DN A
is
nonhomologous
the integration site will influence the gene
(position e f f e c t ) .
hom ologous
1990;
integration
Since
Recently,
re combination
control the integration site
et al.,
the
is a product of an
Lee et al.,
has
gene targeting in
been
at tem pt ed
to
(Paszkowski et a l ., 1988; Baur
1990).
This would be useful in
endogenous plant genes at their natural position
in the genome or in delivering foreign DNA into a predicted
genomic
l o c at io n
to
eliminate
the
possibility
of
the
position effect in the transgene.
Gene
Copy
copy number
number
of
the
can also alter the
introduced
gene
T-DNA
expression.
varies
among
91
transformants.
It has often been
shown that there
is no
positive correlation between increased copies and increased
expression
1989) .
of
tends
introduced gene
(van
genes
Recently,
in sertion
copies
the
Truncation,
krol
the
it has been
to
result
in
et
reduces
a l .,
or
(Shirsat
reported that
1990).
the
1990/
rearrangement
T-DNA
in cr eas ed
(Hobbs et al.,
to the genome
der
in
exp res si on
level of gene
re petition
et
al.,
single T-DNA
Addition
Napoli
et
the
of extra
expression
al.,
of the
of
1990).
introduced
T-D NA may also affect gene expression.
To demonstrate
stable
inheritance
tobacco first generation progeny plants,
of the AFP
show a distinct
in
the progeny plant
DNAs were subjected to Southern blot analysis
The proge ny plants
gene
(Figure 28).
1.1-kb Hindlll
fragment
hybridizing with the 174-bp AFP probe.
5. Kanamycin gene segregation test
Kanamycin gene segregation was observed in the progeny
pl a nt s
(Table
5) .
tra nsgenic plants
seedlings were
re sistance
100
were
scored
(green)
to
or
150
seeds
germinated on
for
from
selective
susceptibility
kanamycin
each
after
of
me di um and
(bleached)
4 weeks.
the
It
and
has
been known that the integrated Ti plasmid is inherited as a
92
Figure
tobacco
stable
28.
Southern
hybridization
first generation progeny plant
inherita nce
of
the
AFP
gene,
analysis
DNAs.
the
of
To confirm
progeny
plant
DNAs were isolated from leaves and digested with Hindlll.
The 174-bp AFP DNA was used as a probe.
Lane
p ro gen y plant,
from transgenic
tobacco p l a n t s .
lanes 2-4:
progeny plants
1: control
93
Table 5. Kanamycin Gene Segregation
number of seedlings
Ratio
KmR
Kms
101
44
LI
142
8
L5
102
48
3:1
L25
114
36
3:1
L2 6
138
10
15:1
L32
118
32
3:1
Gil
94
6
15:1
G13
94
6
15:1
Plants
Control(C2)
(KmR/Kms)
3:1
15:1
94
n o rm al
Mendelian
tr ai t
in
the
progeny
of
transformed
plants.
For plants L5,
the
3:1
gene,
ratio
expected
indicating
Some prog eny
gene
of
plants
the
and L32,
for
a single
s eg re ga tio n
copies
L25,
a
kan a my ci n
single
do mi n an t
copy kanamycin
showed
ratio,
the segregation data fit
a 15:1
gene
resistance
ka nam yc in
in d ic at in g
Me n d e l i a n
the
resistance
presence
s e g r e g at in g
gene.
in
a
of
two
do minant
fashion.
Jorda n
and
tumefaciens
c on t a i n i n g
resistance
McHughen
carrying
bot h
a
was
a
reported
disarmed
ch ime ric
gene was
co-seg re gat io n
(1988b)
NPTII
Ti-plasmid
gene
an d
ob ser ved
in the
pr og en y
likely to be
it may
a
co-segregated
suggest
copy of the kanamycin
A.
vector
gl yp hos at e
for
tissue,
kanamycin
As the NPTII gene and the AFP
gene are located in the same T-DNA region,
Therefore,
when
transf orm ed to flax hypocotyl
and glyphosate resistance.
are most
that
these two genes
in the plant
that plants
containing
gene also have a single
genome.
a single
copy of the
AFP g e n e .
6. Expression of the AFP gene in tobacco plants
The
total
RNA that was isolated from the transgenic
95
t o ba cc o
tissues
probe.
The
inc lud es
was
hybridized
e x pe ct ed
30
mR N A
nucleotides
174-bp of the
AFP DNA,
the AFP gene.
The
are given in figures
with
length
the
is
upstream
about
of
and about
174-bp
400-bp,
the
AFP
abundance
the AFP
of these
D NA probe
was
the
analysis
for the plants transformed
putative
a
which
190 bases dow nstream of
with LB A 44 04/p B 1121AF and GV3111SE/pMON200AF,
The
DNA
gene,
results of the Northern blot
2 9 and 30
AFP
AFP
RNAs
significantly
respectively.
hy br id iz ed
high
amount
with
in the
total RNA extract.
The
the
du pl ic at ed
CaMV35S
transcriptional
duplicated
CaMV
transcriptional
exp ression
of
pr om o te r
activity
35S
the
upstream
e n h an ce rs
foreign
of
for
genes
m ay
indeed
AFP
gene.
sequences
obtaining
hi g h
in tran sg eni c
increase
The
ac t
as
levels
of
plants
(Kay et
a l . , 1987) .
The
CaMV 35S promot er
'constitutive'
1987) , but
genes
(Harpster
recent
fused
to
(Williamson
1989) .
found that
stem,
root,
and
flower
al.,
1988;
suggest
CaMV
constitutive
They
et
studies
the
activity has been
et
35S
tissues
Sanders
the
promoter
al.,
younger,
that
d es cr ibe d as
1989;
con ta in e d
state levels of the RNA than did old tissues.
a l .,
exp ression
of
may
be
Benfey
actively
et
not
et
dividing
hi gh er
al.,
leaf,
steady
LI L13L25L26L30
Figure
RNA
from
29.
Northern
tobacco
LBA4404/pBI121AF.
AFP
gene
at
plants
analysis
of total
transformed
To demonstrate
tr an sc ri pt ion al
transgenic tobacco
the
level,
with
expression
total
RNAs
of the
from
the
leaf tissues were hyb ridized with the
174-bp AFP DNA probe.
with
hybridization
L B A 4 4 0 4 / p B I 121,
Lane 1: control plants transformed
lanes
2-6:
transformed with LB A4 4 04 /p B I 1 2 1 A F .
tobacco
plants
Figure
RNA
from
30.
No rthern hyb ridization
tobacco
GV3111SE/pMON200AF.
tobacco
DNA
plants
Tot al
RNAs
analysis
transformed
from
the
leaf tissues were hy bridized with the
probe.
Lane
GV3111SE/pMON200,
1:
c on tro l
lanes
2-5:
with GV3111SE/pMON2 00AF.
of total
pl a nt s
tobacco
transgenic
174-bp AFP
transformed
plants
with
wi t h
tr an sf or med
98
In plants LI
(lane 2 in figure
29)
and L26(lane
5 in
figure 29) , the high molecular size of RN A hybridized with
AFP DNA probe was also detected in longer exposure
time.
The amounts were significantly lower than the correct gene
size
transcribed.
loci
in
the
kanamycin
indicates that
the
AFP
Since
gene
LI
se gr eg at ion
the mul tiple
into
and L26
the
showed double
test,
insertion
tobac co
this
or
genom e
NPTII
possibily
rearrangement
may
cause
the
of
high
molecular size RNA bands.
The transcripts of the AFP gene were accumulated to a
similar extent
29)
which
in each plant,
showed
one
of
the
except L13
highest
(lane 3 in figure
GUS
activity.
The
level of the AFP transcript of L13 was much lower than that
of the
other
transfo rme d plants.
case the expression
not
correlate
exp ression
plant
of the GUS gene
well.
There
of ph ys ic al ly
genome
can
It
vary
are
seems
and the AFP
reports
linked
genes
independently
that
to
in this
gene
does
show that
c o tr an sf er re d
(Jones
et
the
in
al.,
a
1985;
Nagy et a l ., 1985) .
The
transcription
transformed
significantly.
plants
Such
levels
with
the
AFP
gene
G V 3 1 1 1 S E / p M 0 N 2 0 OAF
differences
could be explained by positional
and gene copy numbers
of
inserted.
in the
effects,
in
the
differed
expression
level
DNA methylation
99
The
mature
antifreeze-protein
expression
in
the
transformed leaf tissues was detected by protein blotting.
Partial
p u ri fi ed
AFP
compared as control.
from
the
winter
fl ounder
serum
was
The antiserum was raised against the
winter flounder AFP peptide 3
(Figure 1),
which has almost
an identical sequence and conformation with IIA7 except
contains
three-11
us ed
this
in
weight
amino
acids
ex p er i me nt
be c a u s e
of
a d di ti on
of
exhibits
more
an
sh ou ld
addition
repeating unit than the
11-
repeating
of
be
of
one
antifreeze
ac id
5.5
more
smallest AFP
ami no
units.
The AFP
kd
11
mo l e c u l a r
ami no
sequence.
repeating
function because
it
acid
One more
unit
p ro b a b l y
of more binding
sites with water molecules in ice lattice.
Figure 31 shows the results of Western blotting.
control
AFP
from
i m m u n o re ac ti ve
-protein
were
winter
band
corresponding
(about 4 k d ) .
iden tif ie d
occurence of
in
flounder
serum
to
The
produced
mature
a
a nt ifr ee ze
The immunoreactive 5„5kd AFP bands
samples of
the doublet
in
LI,
L25,
and
L26.
The
immunoreactive peptides
indicate a specific cleavage or degradation
in the
transformed tobacco
plants.
doublet were almost
equal to each other, the indication
may
be
cleavage
AFP
that
a
specific
stably remained in plants.
Since the
of AFP
may
in
AFP
intensities of the
o cc ure d
and
the
100
1
2
3
4
5
LJ
Figure
31.
Western
from transgenic p l a n t s .
anti fr eez e
prote in
proteins were
run
in
blot
analysis
of
total
to ba cc o
against
the
AFP
Lane
molecular
peptide
The
used
lane 3:
ra ise d
immunoblotting.
lane
control plant
4-8:
total
leaf and
antibody
for
size marker,
L B A 4 4 0 4 / p B I 1 2 1 , lanes
L B A 44 04 /p BI 1 21 AF .
gel.
3 was
weight
flounder serum,
protein
plants,
isolated from transformed tobacco
polyacrylamide
wi t h
8
L25 L 2 6 L 3 0
tran sge ni c
the
winter
7
To demonstrate the expression of
on
1:
6
tr an sg en ic
2:
AFP
from
transformed
plants
with
The arrow indicates the immunoreactive
5.5 kd an tif ree ze - pe p ti de .
101
Th e r e
is
molecular
some
weight
explanations
plant
p ro t e i n
mixtur e
of
th e
as
AFP
absorbed
or
2)
with
tw o
mul ti ple
the
shared by
the
other
con tains
specificities.
the
ty pe
Western
1)
To
antiserum
tobacco
protein
the
existed.
The cold-induced proteins in plants might have a
si gnal
conformation to AFP,
(Kurkela
and Franck,
might
t he
for example,
1990) .
express
but
somewhat
problem
still
the Kinl protein
Therefore,
proteins
It
was
reduced
background
blot.
a
before
plants
the
wild
po ssi ble
are
antiserum
high
ex t rac t
similar
doing
epitopes
the
the
The
binding
p oss ib ili ti es,
with
in
background.
recognizes
antigens,
the se
a
binding
non-specific
an ti bod ie s
distinguish
primarly
ban ds
for
antibody against
non-specific
the
containing
transfo rme d
the
s ha re d
epitopes with the antibody against AFP.
T he
molecular
he ter og en ou s
experiment,
the exact
weight
control
AFP
as well as
calculation
t ra ns for med
tobacco
and
difference
AFP
im mun oreactive
control AFP
Silver
construct
us e d
the non-specific binding,
of the
plants.
level of AFP
However,
expression was highly significant,
the
between
5.5kd
band
was
in
the
this
hampered
accumulation
the
amount
in
of AFP
because the intensity of
much
stronger
than
the
(about 5 ug) .
staining
of
the
total protein showed almost
102
identic al
(Figure
e x p r e ss io n
32) .
identical
pat te r ns
Moreover,
in
the
accumulation
in the transgenic plants
transformed
of
plants
6kd proteins
was
and the control plants,
but the immunoreactive 5.5kd peptides were
identified only
in the transfo rm ed plants,
control
This
undoubtly
and not
eliminates
specific bi nd in g
of the
5.5
the
in the
possibility
kd pept ide
of
with
the
plants.
the
non
antibody
against AFP.
7.
Frost
tolerance
of
seedlings
in
the
transgenic plants
The frost tolerance of tobacco seedlings
and
transgenic
determined.
were
plants
c o n t a i n i n g th e
from control
AFP
ge ne
was
The seedlings which were kanamycin resistance
selected,
tr ansferred to Jiffy
soil,
and grown
in an
environmental growth chamber.
The plants
the
were
pl ac ed
p o s i t i o n i n g effect
controlled
incubator.
multisensor
temperature
fr ee zin g
the
chamber,
of
plants
Four
a
the
to
distributed
than
eliminate
temperature
thermocouples
less
at the time
0°C
in
recorder,
indicated
air temperature
randomly at
0 . 5°C
from
into
spread
a
the
in
of m i ni mu m temperature.
103
2
3
4
5
kd
29.5
18-7
15.5
5.9
2.9
Fi gure
transgenic
-
32.
—
O
H
)
*
ft
V
Silver
pl a n t s .
tr ans fo rm ed tobacco
gel.
Lane
1:
staining
Total
leaf were
m ol e c u l a r
of
total
proteins
run
weight
on
p r ot ei n
isolated
from
from
the po ly acrylamide
size
marker,
control plant transformed with LBA4404/pBI121,
transgenic plants with LBA 44 0 4/ pB I1 21 AF .
lane
2:
lanes 3-5:
104
The
results
control
are
is
shown
the
in
Table
transformed
6 and
p lan ts
figure
without
33.
the
The
AFP
gene
coding s e q u e n c e .
An increase in frost tolerance compared to the control
plants was observed in plants L25 and L26,
well
wi th
the
resul t
preliminary test,
which has
been
of
the
Western
the
reported by Lindow et
result
tra n sg en ic
Only
some
killing
and
of
largest
cm)
of
a
chilling
control,
the
leaf
size
showed more
In
plants
the
about
All
5-6
plants,
k il led
at
depending
of
and this
sustained at -7°C was
s u r vi ve d
The plants
was
(1982),
co mp l et el y
varied
stage.
al
injury.
were
small
temperature
developmental
blot .
the tobacco plants had no damage at -5°C,
indicates that the damage the plants
not
which coincides
on
at
both
-8°C.
-8°C.
The
th e
pl a nt
leaf stage
15 m m and height
was
(the
about
2
freezing tolerance than the bigger plants
(height ~8 c m ) .
As
described previously,
is induced at
AFP
(Kurkela
vitr o
cold stress,
and
application
inc r ea se
Therefore,
the
the
location may
Franck,
of
the
has
tolerance
high
expression
attribute
the
(Cutler
of
the
which
composition to the
In
in plant
frost
gene product
similar
1990).
the AFP
kinl
addition,
leaves
et
AFP
does
al.,
in
the
in
indeed
1989) .
cytoplasmic
frost tolerance in plants L25
105
Table
6.
E n h a nc em e nt
The
plants
for
frost
which
of
were
tolerance
frost
tol er an ce
kanamycin
in the
of
resistance
freezing
seedlings.
were
chamber.
(%) indicates the mean value of three replicates.
Plants
na
Survival
Control
24
35
LI
21
48
L5
24
35
L25
22
55
L2 6
20
56
a: Number of plants tested
(%)
te sted
Survival
C 2
Fi g u r e
plants.
33.
JL 25
C o m p ar i so n
L 26
of
f re e z i n g
tested
to ba cc o
C2 indicates control plants without the AFP gene
coding sequence and the front row indicates half freezing
damage d plants.
freezing at -7°C.
The
picture
was
taken
two
weeks
after
107
and L26.
However,
the plant L5 did not show any frost tolerance
in the latest freezing test
some
frost
later
tolera nc e
f r ee zi ng
kanamycin
that
germination
some
L5
L5
may
p r oc e ss
expression.
Therefore,
test,
the
The
suffer
have
some
the
integration
ma y
test.
very
poorly
in
lost
tests,
This
in
the
seed
al t e r a t i o n
of
the
gene
site of AFP
effects
in
the
seed
AFP
gene
in L5 may
ger mi nation.
gene
during
seed
In the
L5 ge rminated very well,
95% like that of other plants.
the
changes
germination or during the period of seed storage.
previous pr el i mi na ry
In the
(about 30% g e r m i n a t i o n ) .
ca us in g
deleterious
L5
p r e l im in ar y
germinated
selection plates
indic at es
cause
in
(Table 6) even though it showed
over
During the time passed,
L5
lost some of its germination ability,
and may have lost the
AFP
L5
gene.
This
could
explain
survival as the control plants.
why
has
same
level
of
108
Summary
In
this
dissertation,
construction
and
an tifreeze-protein
plants,
transformation
gene
from winter
of
flounder
the
plasmid
the
mature
into
tobacco
To maximize
the expression
of the mature AFP
a duplicated CaMV 35S promoter was used instead of a
single
using
CaMV 35S promoter.
the
double
CaMV
si g ni fic an tly
highe r
Since
gene
have
describe
and the frost tolerance of these transgenic tobacco
seedlings.
gene,
I
the
AFP
also
affect
The
level of AFP transcription
35S pr omo te r
than
has
AFP
the
a high
gene
p la sm id
single
GC
Ca MV
35S
content
expression
construct
was
promoter.
(~ 80%) , it may
by
serving
as
DNA
methylation sites or stabilizing the mRNA.
The preliminary freezing tests of transgenic seedlings
a nd
analysis
transgenic
phy sically
of
p lan ts
transgenic
not
have
gene.
expression
were
linked genes
the
to
AFP
plants,
correlate
by
Western
p er fo rm ed.
can be
a high
with
a
Plants LI, L5, L25,
This
expressed
level
high
of
blots
was
of
b e ca us e
independently
GUS
activity
expression
in
does
of the AFP
and L26 were chosen for further
freezing tests.
The
plants
exp ression
does
transgenic
confer
plants
of
the
frost
an tif r ee ze -p ro te i n
tolerance.
survived
freezing
At
in
least
tobacco
30%
more
conditions than the
109
control
the
plants.
AFP
gene
cytoplasmic
These
was
results
designed
location,
indicate
to
be
that
even
though
expressed
a high-label of AFP
in
a
expression can
prevent freezing injury.
Since ice formation occurs
regions
the
of
the
plant
cells,
ant ifr eeze-protein
one
to be
hypothesis
would
freezing
However,
this rational is somewhat c o n t ro v er sa l.
caused
by
require
expressed ex tracellularly to
prevent
dehydration
injury
first at the extracellular
caused
by
cellular
extracel lu lar
lethal at the time of freezing.
ice
dehydration.
Cellular
formation
Otherwise,
is
not
intracellular
ice formation which occurs at rapid cooling rates is lethal
in
p la nts .
Th er ef ore ,
an t if re ez e - p r o t e i n
intracellular
ice
ma y
have
already
protein,
ma y
cytoplasmic
reduce
cell
formation during
such
a
damage
by
of
The
an
pr ev e nt in g
rapid cooling.
protein.
the kinl gene product,
expression
Plants
cold-induced
is a good example.
Targeting of an antifreeze-protein into a cell vacuole
may
impr ove
contains
vacuole
the
most
also
degrade
by plants.
problems
of
frost
water
contains
in the plant cell.
varieties
the metabolites
Therefore,
in
and
of
the
v ac uol e
However,
hydrolytic
enzymes
the
to
abnormal proteins produced
vacuole targeting may face several
designing
ant ifreeze-protein
tolerance, b e c a u s e
without
the
expression
degradation
in that
of
the
organelle.
110
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VITA
Jung-Sook
Youn g -J a
August
S.,
28,
Food
Lee.
1958.
graduated
in
Lee
is
th e
daughter
She
was
born
University
in
in
Won-Sub
Pusan,
Korea,
She attended Nam-Sung High School
in February
Science
of
and
1977.
She obtained a B.S.
Tec hnology
February
1981.
from National
She
be g an
and
on
and
Degree
Fisheries
her
graduate
study in the Department of Food Science and Technology at
the
Se o u l
National
University
in
March
graduated with a M.S.
Degree in February 1983.
her
the
Ph.D
program
at
Depa rtm en t
of
1981,
and
She began
B i o c h e mi s tr y
in
Louisiana State University in June 1985.
Sbe is married to Joon-Ho Lee and they have a son,
Min-Sang.
of
Currently,
Philosophy
at
the
she
is a candidate
Department
Louisiana State University,
of
Baton Rouge,
for the Doctor
Biochemistry
Louisiana.
in
DOCTORAL EXAMINATION AND DISSERTATION REPORT
Candidate:
Major Field:
Jung-Sook Lee
Biochemistry
Title of Dissertation:
The frost tolerance of tobacco plants transformed with
the gene encoding the antifreeze-protein from winter flounder
Approved:
M ajor Professor and 'C hairm an
Dean of the G raduate School
EXAMINING COMMITTEE:
-
Date of Examination:
July 11, 1991