Expression of insect (Microdera puntipennis dzungarica) antifreeze
Download
Report
Transcript Expression of insect (Microdera puntipennis dzungarica) antifreeze
Antifree proteins (AFPs) have been
isolated from several organisms.
› Fish, insects, plants, bacteria
Bind to ice crystals, inhibit growth, lower
freezing point and not melting point.
Thermal hysteresis activity (THA)difference between melting point and
nonequilibrium freezing point.
Used as an indicator of AFPs activity, so
AFPs often referred to as thermal
hysteresis proteins (THPs).
Varies among species: Insects (3-6 ºC),
Fish (0.7-1.5 ºC), Plants (0.2-0.5 ºC)
AFP-producing insects: goal is to avoid
freezing, cannot survive if body fluids
actually freeze.
AFPs lower freezing point of hemolymph
and gut fluid to prevent freezing from the
external ice across the body surface.
Can achieve higher crop yields by
improving freezing tolerance of plants.
Therefore, want to express AFPs in frostsusceptible crops to increase their cold
tolerance.
Some of the most effective ATPs found in
insects.
Want to test the MpAFP149 gene,
isolated from the beetle Microdera
puntipennis dzungarica,in its ability to
increase cold-tolerance of transgenic
tobacco plants and protect them from
freezing damage.
363 bp with a signal peptide sequence
Transcript encoding 98 amino acids of
mature peptide is 68.37% homologous
with published AFP from Tenebrio molitor
(Tm)
Tm expressed successfully in E. coli; high
activity at protecting bacteria at low
temperature, linearly correlated with AFP
concentration.
Confirm expression of MpAFP149 in
plants and visualize sub-cellular
localization
MpAFP149 gene with 35S promoter fused
with green fluorescent protein (GFP) in
plasmid pCAMBIA1302-GFP
Introduced into onion epidermal cells via
particle bombardment
MpAFP149 gene with signal peptide
sequence obtained by PCR.
Gene construct
CaMV35S-MpAFP149-Nos inserted into
plasmid pCAMBIA1302 with HindIII and
EcoRI to form expression vector
pCAMBIA1302-MpAFP149
Expression vector pCAMBIA1302MpAFP149 transferred into competent
Agrobacterium cells (EHA105 strain)
DNA extracted from kanamycin-resistant
surviving Agrobacterium colonies
PCR to confirm presence of MpAFP149
transgene
1-2 in. young tobacco leaf discs infected
with EHA105 Agrobacterium containing
pCAMBIA1301-MpAFP149.
Cultivated in the dark at 28 ºC for 2 days.
Leaf discs transferred to generation
medium supplemented with hygromycin.
T0 plants allowed to grow and flower
and set seeds in a growth chamber.
Allowed to grow 15 weeks in green
house before harvesting seed capsules
T0 and Wild-type seeds sterilized by
soaking in 1:9 (v/v) 30% bleach:ethanol
Rinsed 5 times with ethanol and left
overnight to volatize ethanol
T0 seeds germinated on plates with
hygromycin to select for seedlings
carrying HPTII gene
Transplanted into pots to full growth at
25 ºC
Extracted genomic DNA and performed
PCR to identify MpAFP149 gene
RNA isolated from plant leaves and
reverse transcription carried out
RT-PCR products run through agarose gel
electrophoresis to check MpAFP140
transcription
Wild-type leaves and transgenic
tobacco leaves
Polyclonal antibody raised in mouse
against MpAFP149 protein
Immunogold labeling (Antibodies
conjugated to gold particles)
Extracted apoplastic proteins from
leaves and separated by sodium
dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE)
Western blot with antibody against
MpAFP149 protein
After growing for one month, three
transgenic and three wild-type plants of
similar growth states and no visible
phenotypical differences were chosen
to undergo cold treatment, measure
electrolyte leakage, and
Malondialdehyde (MDA) content.
Set temperature in freezing chamber to -1
ºC for 0, 24, 48, and 72 hours and observed
phentypes.
In addition, leaf samples from each group
were washed with deionized water and
then immersed in deionized water.
After vacuum infiltration, the electric
conductivity of supernatant was detected.
MDA- natural occuring reactive species
that is a marker for oxidative stress
The level of MDA at -1 ºC was
determined to analyze the comparative
rate of lipid peroxidation.
The localization of MpAFP149 was
determined by expressing
MpAFP149:GFP construct plasmid in
onion epidermal cells.
For the control, fluorescence was seen
throughout the entire cell and for the
transformed cells it was solely in the
apoplast (see Figure next slide).
35S-MpAFP149-NOS vector transformed
into tobacco using Agrobacteriummediated gene transfer.
Screened by hygromycin and tested for
the presence of the vector by PCR.
Two samples, T0-5 and T0-39 showed
higher transcript level by RT-PCR.
These two lines were chosen for detailed
analysis.
Immunogold labeling approach used to
determine if MpAFP149 protein was
expressed and where it localized in
transgenic tobacco.
Showed that MpAFP149 protein
accumulated in outer layers of cell wall
in transgenic plant, but absent in control
tobacco plant
Western blot for apoplastic proteins
showed expected protein band of 10.2
kDa, indicating that mature peptide
protein MpAFP149 synthesized in
transgenic tobacco.
When exposed for 1 day, both
transgenic and wild-type tobacco plants
only exhibited moderate dehydration.
When exposed for 2 and 3 days, most
leaves of wild-type were frozen but
transgenic tobacco only exhibited
dehydration of a few older leaves near
the plant base.
After returning to room temperature,
MpAFP149 plants overcame dehydration
and recovered completely.
Wild-type displayed permanent
damage.
Transgenic line displayed improved cold
tolerance and enhanced recovery
Low temperatures disrupted semipermeability of tobacco
cytomembranes
Effusion of electrolytes resulted in
increased electrical activity of tissues
Over time, ion leakage difference
increased between control and
transgenic tobacco.
Increase of MDA parallels the increase in
conductivity/ion leakage, one does not
cause the other.
Wild-type plants suffered higher
oxidative lipid injury than transgenic
plants; correlated to increases in ion
leakage and MDA content.
Transgenic tobacco plants expressing
MpAFP149 protein with the signal
peptide showed improved tolerance to
cold and an enhanced recovery.
MpAFP149 may be used as a candiate
for the improvement of frost-resistant
crops.
Wang, Y., Qiu, L., Dai, C., Wang, J., Luo,
J., Zhang, F., & Ma, J. (2008) Expression of
insect (Microdera puntipennis
dzungarica) antifreeze protein
MpAFP149 confers the cold tolerance to
transgenic tobacco. Plant Cell Rep 27:
1349-1358.