CN106119260B - The huge peak adversity gene VlbZIP36 of America and Europe's hybridization grape variety and its application - Google Patents
The huge peak adversity gene VlbZIP36 of America and Europe's hybridization grape variety and its application Download PDFInfo
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Abstract
A kind of huge peak adversity gene VlbZIP36 of American-European hybridization grape variety and its application are disclosed in the present invention.Disclosed gene entire open reading frame sequence 969bp, encodes 322 amino acid.Inventor has found that the transgenic arabidopsis of overexpression VlbZIP36 showed to enhance the resistance to drought stress in Their Seed Germinating Period, Seedling Stage, maturity period, and the resistance is related with the expression enhancing of the change of relative conductivity and malonaldehyde and stress-related genes.VlbZIP36 gene is to regulate and control the activity of antioxidase by limiting loss of moist to remove ROS, the expression of transcriptional control related objective gene plays the role of enhancing drought stress resistance on transgenic arabidopsis.Disclosed application is the application for the drought resisting stress ability that gene VlbZIP36 is used to improve plant.
Description
Technical field
The present invention relates to plant stress-resistance identified for genes and gene engineering technology field, in particular to one American-European hybridization grape
The huge peak adversity gene VlbZIP36 of kind.
Background technique
Basic leucine zipper bZIP transcription factor is that most extensive, most conservative one kind is distributed in eucaryote transcription factor
Albumen is found in people, animal, plant, microorganism and insect.The bZIP transcription factor phase in various plants at present
After being found, for example, there are 75 in arabidopsis gene group, there are 89 or 92 in rice genome, 47 is found in soybean, sorghum
Middle discovery 92 finds 125 in corn, discovery 47 or 55 etc. in grape genome.According to the similar of their structural domain
Property and conservative, can be divided into 10 subfamilies, i.e. A, B, C, D, E, F, G, H, I, S；Or 13 subfamilies, i.e. A, B, C, D,
E, F, G, H, I, J, K, L, S etc..
Plant bZIP transcription factor is structurally characterized in that: (1) peptide chain C-terminal includes a highly conserved bZIP structure
Domain, structural domain be by DNA integrated structure area and leucine zipper structure district's groups at.Leucine zipper region and DNA integrated structure
Area is closely coupled, and every 7 amino acid just includes a leucine.Leucine zipper forms the helical structure of an amphiphilic, should
Structure participate in bZIP albumen in conjunction with DNA before it is Dimerized；(2) N-terminal of transcription factor contains acid activatable area；(3)
After forming dimeric forms, DNA integrated structure area and the DNA of peptide chain C-terminal are bound directly.In addition, other than bZIP structure domain,
There are also some other conserved domains, such as proline to be rich in domain and acidic amino acid structural domain rich in domain, glutamine, this
A little structural domains may have the function of transcriptional activation.
Grape is to cultivate one of widest fruit tree in the world, has important edible value and economic value.But due to
It is often exposed in various biologies and abiotic stress, seriously affects it in the yield and quality of big Tanaka.Cultivation in production
Grape variety belongs to European grape mostly, has the characteristics that resistance is poor, but the amur grape in China is not only degeneration-resistant, disease resistance
It is relatively strong and preferable with European grape cross compatibility, effectively resistant gene and European grape quality trait mutually can be tied
It closes to carry out the genetic improvement of Grape Germplasm resource.Plant vital movement rule under poor environment is studied, its resistance is disclosed
Physiological foundation and molecular mechanism, not only facilitate fully understand plant normal physiological activity, and to cultivate have resist
The excellent variety of poor environment character has important directive function.
Summary of the invention
The object of the present invention is to provide an American-European hybridization huge peak adversity gene VlbZIP36 of grape variety, and prove
Function of the gene at degeneration-resistant aspect.
In order to realize that above-mentioned task, the present invention use following technical solution:
A kind of American-European hybridization huge peak adversity gene VlbZIP36 of grape variety, which is characterized in that the America and Europe hybridizes grape product
The coding region sequence of the huge peak adversity gene VlbZIP36 of kind is as follows:
The present invention constructs pCAMBIA2300-35S-VlbZIP36 Overexpression vector for the first time, and passes through titbit dip method
It is conducted into model plant arabidopsis.Have studied turning for the American-European hybridization huge peak adversity gene VlbZIP36 overexpression of grape variety
Gene strain and wild to impinging upon osmotic stress, dehydration processing, the growing state under prolonged drought Stress treatment.
The American-European hybridization huge peak adversity gene VlbZIP36 of grape variety of the invention can significantly improve arabidopsis for infiltration
The tolerance of stress, dehydration processing and prolonged drought stress.VlbZIP36 gene of the invention is adjusted by limiting loss of moist
The activity of control antioxidase is to remove ROS, the expression of transcriptional control ABA and stress-related genes, in the quasi- south of transgenosis
Play the role of enhancing drought stress resistance on mustard.
Inventor's discovery transgenic arabidopsis strain compared with wild type shows the resistance of enhancing desiccation stress.And this is anti-
Property enhancing show that transgenic line moisture holds the enhancing (reduce percentage of water loss, less blade here wither) and cellular damage of power
The mitigation (reducing cell mortality, lower conductivity and mda content) of degree.Result above all shows wild Vitis quinquangularis
Overexpression of -24 adversity gene VqbZIP39 of quotient in arabidopsis improves the ability of the drought resisting stress of plant.
Detailed description of the invention
Fig. 1 is VlbZIP36 compared with other plant bZIP amino acid sequence；Wherein: A figure is VlbZIP36 and other plants
The phylogenetic analysis of object bZIP amino acid sequence；B figure is the highly conserved amino acid residue sequence of VlbZIP36 and other plants
The amino acid residue sequence Multiple sequence alignments of bZIP in object, wherein the Genbank accession number of related gene is AtbZIP60
(NP_174998.1), OsbZIP74 (XP_015641141.1), AtABF2 (NP_001185157.1), AtABF3 (NP_
849490.2), OsbZIP10 (XP_015650926.1), OsbZIP11 (XP_015625853.1), AtABF1 (NP_
564551.1), AtABF4 (NP_566629.1), AtABI5 (NP_565840.1), OsABF1 (XP_015612904.1) and
OsbZIP14(XP_015622690.1)。
Fig. 2 is expression pattern situation of the VlbZIP36 promoter in arabidopsis T3 for different stages of growth in plant；A figure is
24 hours mature embryos of MS culture medium are grown in, scale is 200 μm；B figure is to be grown in the MS culture medium containing 300mM mannitol
48 hours mature embryos, scale are 200 μm；C figure is the seedling in 5 day age, and scale is 500 μm；D figure is the young plant of 2 week old, scale
For 2mm；E figure be grown in the MS culture medium containing 300mM mannitol 7 days 2 week old young plant, scale 2mm；F figure is 3
The young plant of week old；G figure is 3 week old young plants after dehydration is handled 2 hours；H figure is 3 week old after being handled 1 hour with 10 μM of ABA
Young plant, scale 2mm；I figure is the guard cell of 3 week old young plants, and scale is 50 μm；J figure is 3 weeks after dehydration is handled 2 hours
The guard cell of age young plant, scale are 50 μm；K figure is the guard cell of 3 week old young plants after being handled 1 hour with 10 μM of ABA,
Scale is 50 μm；L figure is titbit, scale 2mm；M figure is flower, and scale is 200 μm；N figure is the style and anther of flower, and scale is
200μm；O figure is silique, scale 1mm.
Fig. 3 is the seed of wild type and VlbZIP36 transgenic arabidopsis strain (13#, 31# and 32#) under osmotic stress
Sprouting situation；A figure is wild type and transgenic line seed in MS culture medium, add the MS culture medium of 350mM mannitol on train
Morphology picture after supporting 3 days；B figure is wild type and transgenic seed in MS culture medium, addition 300,325 and 350mM sweet dew
Germination percentage situation after being cultivated 3 days on the MS culture medium of alcohol；* indicate that there were significant differences compared with wild control, * * is indicated and open country
Raw control, which is compared, extremely significant difference (P < 0.01 * 0.01 < P < 0.05, * *).
Fig. 4 is the cotyledon of wild type and VlbZIP36 transgenic arabidopsis strain (13#, 31# and 32#) under osmotic stress
Afforest situation；A figure is wild type and transgenic line seed in MS basal medium, addition 300,325,350mM mannitol
Cotyledon after cultivating 6 days on MS culture medium afforests picture；B figure is that wild type and transgenic line tie up to MS basal medium, addition
300, the cotyledon greening situation after 325, being cultivated 6 days on the MS culture medium of 350mM mannitol；C figure is wild type and transgenic line
It ties up to MS basal medium, add the cotyledon greening situation of different growth times on the MS culture medium of 350mM mannitol；* it indicates
There were significant differences compared with wild control, * * expression have compared with wild control extremely significant difference (* 0.01 < P < 0.05, * * P <
0.01)。
Fig. 5 be compared with wild type VlbZIP36 transgenic arabidopsis strain (13#, 31# and 32#) growth period after germination
Between enhancing to the resistance of osmotic stress；A figure is that wild type and transgenic seedlings are sweet in MS basal medium, addition 300,350mM
Reveal the morphology picture after cultivating 7 days on the MS culture medium of alcohol；B figure be wild type and transgenic seedlings MS basal medium,
Add the root long statistical conditions after cultivating 7 days on the MS culture medium of 300,350mM mannitol；C, D figure is the wild type of a week old
It is cultivated from the MS culture medium for being transferred to MS culture medium, addition various concentration mannitol in MS basal medium with transgenic line
Relative conductivity (C) and mda content (D) after 7 days；E figure is that wild type and transgenic line tie up to and carry out Osmotic treatment in soil
Morphology picture and statistics survival rate；DDT represents the number of days of Osmotic treatment.* indicate there is significance difference compared with wild control
Different, * * expression has extremely significant difference (P < 0.01 * 0.01 < P < 0.05, * *) compared with wild control.
Fig. 6 is that VlbZIP36 transgenic arabidopsis strain (13#, 31# and 32#) can enhance arabidopsis pair compared with wild type
The resistance of Water deficit；A figure is 3 week old wild types and transgenic line ties up to the morphology before dehydration and after dehydration 1,3,6 hour
Picture；B figure is that the blade tied up to before dehydration and after dehydration 6 hours to wild type and transgenic line carries out Trypan Blue situation；C
Figure is the percentage of water loss statistical conditions of wild type and transgenic line during dehydration 6 hours；D, E figure be 3 week old wild types and
Transgenic line ties up to before dehydration and the relative conductivity (D) and mda content (E) of 6 hours rear blades of dehydration；F figure is 3 week old
Wild type and transgenic line tie up to the stomata under 10 μM of exogenous aba treatments and close situation, and arrow instruction is guard cell；Scale
=50 μm；* indicate that there were significant differences compared with wild control, * * expression has extremely significant difference (* 0.01 < P compared with wild control
<0.05,**P<0.01)。
Fig. 7 is the reactive oxygen species and antioxygen of wild type and VlbZIP36 transgenic Arabidopsis plants (13#, 31# and 32#)
Change enzyme activity assay；A, B figure is the blade tied up to before dehydration and after dehydration 6 hours to 3 week old wild types and transgenic line, is carried out
NBT (A) and the dyeing of DAB (B) histochemical method, detect O respectively2 -And H2O2Content；C-E figure is before dehydration and dehydration 6 is small
The activity of SOD (C), CAT (D), POD (E) in Shi Hou, wild type and rotaring gene plant blade；* indicate have compared with wild control
Significant difference, * * expression have extremely significant difference (P < 0.01 * 0.01 < P < 0.05, * *) compared with wild control.
Fig. 8 be stress-related genes wild control and transgenic line VlbZIP36 transgenic Arabidopsis plants (13#,
31# and 32#) in expression；After dehydration handles young plant 1 hour and 2 hours of 3 week old, acquisition blade proposes RNA and reverse transcription
Stress gene expression quantity is measured using Real-time quantitative PCR afterwards；Reference gene is Actin2.* it indicates compared with wild control
There were significant differences, and * * expression has extremely significant difference (P < 0.01 * 0.01 < P < 0.05, * *) compared with wild control.
Below in conjunction with drawings and examples, present invention is further described in detail.
Specific embodiment
Largely research shows that bZIP transcription factor family has many important biological functions, including the storage of seeds base
The expression of cause, the growth and development of plant, light signal transduction, disease defence, the sensibility etc. of abiotic stress response and ABA are each
The reaction of kind signal, but the functional study about grape bZIP gene is also seldom.Overexpression is found to the research of arabidopsis
The repellence of plants against abiotic stress can be improved in bZIP, this is of great importance to the improvement of plant resistance to environment stress.Seminar is preceding
Have been found that grape transcription factor bZIP gene has important role in terms of resisting abiotic stress in phase research.Therefore, originally
BZIP family gene VlbZIP36 is cloned in research from American-European hybrid grape (V.labrusca × V.vinifera) ' huge peak ', and
Successful conversion model plant arabidopsis, further the function of research bZIP gene and its possible mechanism of action, are grape drought resisting
Breeding provides theoretical foundation.
The Overexpression vector for carrying the gene coding region VlbZIP36 is transferred in arabidopsis by this research, and analysis transgenosis is quasi-
Response of southern mustard under the conditions of osmotic stress.Under the mannitol osmotic stress of 300,325 or 350mM, transgenic arabidopsis
Percentage of seedgermination and the equal conspicuousness of cotyledon green percentage are higher than wild type.In the mannitol osmotic stress condition of 300 or 350mM
Under, also conspicuousness is longer than wild type to the root long of transgenic line.In summary results presumption is in mannitol osmotic stress condition
Under, the percentage of seedgermination and root growth of VlbZIP36 transgenic arabidopsis are all enhanced.
Osmotic stress can cause membrane lipid peroxidatio, accumulation and the change intracorporal electrolyte of plant so as to cause malonaldehyde
It is horizontal.Therefore mda content and electrolytic leakage are used to assess the journey of abiotic stress resistance as effective index
Degree.In our current research, inventor to 300 or 350mM treatment with mannitol seedling after a week and dehydration handle 6 hours at age
It is found when seedling leaf measurement mda content and level of conductivity, the equal conspicuousness of relative conductivity and mda content of wild type
Higher than transgenic line, show the degree of transgenic line damaged membrane lower than wild type.In addition it originally researchs and analyses
Transgenic arabidopsis strain is at age seedling by the situation after drought stress.It was found that transgenic line ties up to compared with wild control
Arid showed higher drought resistance after 8 days.ABA can promote stomata to close, to reduce the loss of water transpiration.And mesh
It is preceding many research shows that the plant for having preferable resistance to drought stress is often sensitive to ABA, therefore this research is to transgenosis
Strain has carried out the test of ABA sensibility.The result shows that after being handled blade 1 hour with 10 μM of ABA, transgenic line and wild
The stomatal aperture of type is reduced, but the former reduction is more.Therefore inventor speculates that transgenic line can enhance to arid
Perhaps, the resistance of stress is attributed to its sensibility to ABA.
Plant will lead to cylinder accumulation active oxygen under abiotic stress, such as hydrogen peroxide (H2O2), superoxides yin from
Son (O2 -) and hydroxyl (- OH) etc..And these extra active oxygens meeting oxidative cell components, this is considered as that plant cell is impaired
Principal element maintains oxygen balance in plant so removing excessive ROS in time, for improving stress resistance of plant with important
Effect.In this research, inventor has carried out the enzyme activity assay of active oxygen scavenger to the Arabidopsis plant that dehydration is handled, knot
Fruit shows after dehydration 6 hours, the O in wild type2 -And H2O2Accumulation be above transgenosis.In order to reduce active oxygen pair
The destructive power of cell, plant can generate antioxidant and active oxygen scavenger, such as superoxide dismutase (SOD), hydrogen peroxide
Enzyme (CAT) and peroxidase (POD).This research also determines the work of three above antioxidase in plant after dehydration is handled
Property.The result shows that the activity of SOD, CAT and POD are obviously higher than wild type in transgenic line after dehydration 6 hours, show
Transgenic line possesses more effective SCAVENGING SYSTEM OF ACTIVATED OXYGEN than wild type.
Inventor utilizes Homology-based cloning, uses reverse transcription PCR according to European grape PINOT NOIR genome sequence
(Reverse Transcription-Polymerase Chain Reaction, RT-PCR), it is huge with America and Europe's hybridization grape variety
It is template that peak blade total serum IgE reverse transcription, which synthesizes the first chain of cDNA, has expanded the American-European huge peak adversity gene of hybridization grape variety for the first time
VlbZIP36, gene entire open reading frame sequence 969bp, encodes 322 amino acid.
The phyletic evolution of VlbZIP36 amino acid sequence and the bZIP amino acid sequence of other plant A and K groups studied
Tree analysis and its highly conserved amino acid residue Multiple sequence alignments the result shows that, VlbZIP36 amino acid sequence and arabidopsis
AtbZIP60 and rice Os bZIP74 amino acid sequence homology highest, and they belong to the K group subfamily in bZIP family.
VlbZIP36 and other bZIP amino acid structures are analyzed, as a result, it has been found that in their amino acid sequence all comprising one by
The bZIP conserved domain of DNA binding structural domain and leucine zipper motif composition.
In expression pattern analysis test, inventor has found the promoter seed of VlbZIP36 under normal growing conditions
Have in germination period, Seedling Stage, adult seedling stage (in addition to having higher expression in anther, style, silique) and guard cell fainter
Expression, however arid and ABA processing different times young plant after, the active conspicuousness of the gene promoter increases.
In order to which the American-European hybridization huge peak adversity gene VlbZIP36 of grape variety of a step research is resisted in drought stress in plant
Concrete function, inventor constructs pCAMBIA2300-35S-VlbZIP36 (restriction enzyme site be XbaI and KpnI) overexpression
Carrier, overexpression, response of analysis transgenic arabidopsis under the conditions of osmotic stress in wild Arabidopsis plant by it.Hair
Now have the strain of the American-European hybridization huge peak adversity gene VlbZIP36 of grape variety under the mannitol osmotic stress of various concentration
Percentage of seedgermination and cotyledon green percentage equal conspicuousness be higher than wild type.And under identical stress conditions, transgenic line
Also conspicuousness is longer than wild type to the root long of system.In summary results presumption is under the conditions of mannitol osmotic stress, VlbZIP36
The percentage of seedgermination and root growth of transgenic arabidopsis are all enhanced.
In order to further appreciate that mechanism of action of the VlbZIP36 in terms of Osmotic Stress Tolerance, inventor is had detected different dense
The relative conductivity and mda content of wild type and transgenic line under the conditions of the mannitol osmotic stress of degree.The result shows that
After osmotic stress, the equal conspicuousness of relative conductivity and mda content of wild type is higher than transgenic line, shows transgenosis
The impaired degree of lineage cells film is lower than wild type.Furthermore inventor is also by VlbZIP36 transgenic line and the whole strain of wild type
Plant is placed on clean filter paper, simulating drought experiment.As a result, it has been found that transgenic arabidopsis strain shows compared with wild type
Enhance the resistance of desiccation stress out.And the enhancing of the resistance shows that transgenic line moisture holds enhancing (the reduction dehydration of power
Here rate, less blade wither) and cellular damage degree mitigation (reduce cell mortality, lower conductivity and malonaldehyde contain
Amount).ABA can promote stomata to close, to reduce the loss of water transpiration.Therefore inventor carries out transgenic line
The test of ABA sensibility.The result shows that the stomata of transgenic line and wild type is opened after being handled blade 1 hour with 10 μM of ABA
Degree is reduced, but the former reduction is more.Therefore inventor speculates that transgenic line can enhance the resistance to drought stress
Perhaps due to its sensibility to ABA.Furthermore inventor also carries out the method point of chemical staining to dehydration treated plant
Ultra-oxygen anion free radical (O in plant is not had detected2 .-) and hydrogen peroxide (H2O2) level and analyze active oxygen therein
Scavenger superoxide dismutase (SOD), the enzymatic activity of catalase (CAT) and peroxidase (POD).The result shows that
After dehydration is handled 6 hours, accumulated active oxygen is less in transgenic line compared with wild control, and the oxidative stress received is also smaller,
The enzymatic activity of three kinds of active oxygen scavengers is higher simultaneously, illustrates the mistake of the American-European hybridization huge peak adversity gene VlbZIP36 of grape variety
Amount expression may take part in the reset procedure of ROS to enhance the anti-stress ability of plant.
It is the coding region sequence and drought resisting stress function of the American-European hybridization huge peak adversity gene VlbZIP36 of grape variety below
The specific steps of energy experimental verification.
A, in early-stage study analysis, expression of 47 bZIP family genes after Different stress processing and HORMONE TREATMENT
On the basis of, using Homology-based cloning, it is with huge peak blade total serum IgE reverse transcription synthesis the first chain of cDNA of America and Europe's hybridization grape variety
Template, amplification have obtained the huge peak adversity gene VlbZIP36 sequence of American-European hybridization grape variety, and it is huge which hybridizes grape variety
The coding region sequence of peak adversity gene VlbZIP36 is as follows:
B, the entire open reading frame of the American-European hybridization huge peak adversity gene VlbZIP36 sequence of grape variety is inserted into
CaMV35S promoter downstream, construct plant Overexpression vector and by its by the titbit dip method of mediated by agriculture bacillus by its
Import wildtype Arabidopsis thaliana Colombia C0.Screening obtain the good VlbZIP36 transgenic line of phenotype (13#, 31# and
32#).The promoter sequence in 1,973bp of upstream from start codon has been cloned simultaneously, constructs ProVlbZIP36: GUS expression carries
It is simultaneously conducted into wildtype Arabidopsis thaliana Colombia C0 by the titbit dip method of mediated by agriculture bacillus by body.Screening obtains
The higher T3 of GUS expression quantity is for plant.
C, referring to Fig. 3-8, inventor identify VlbZIP36 transgenic line (13#, 31# and 32#) can significantly improve it is quasi-
Southern mustard is for osmotic stress, dehydration processing, the tolerance of prolonged drought.In addition, adding the sweet of various concentration in MS culture medium
Reveal alcohol (Mannitol), the germination rate and cotyledon green percentage of VlbZIP36 transgenic line (13#, 31# and 32#) are all remarkably higher than
The growing way of wild control and transgenic line root is also relatively better than wild type.Under the conditions of Water deficit, in transgenic line
The accumulation of relative conductivity, mda content and active oxygen (ROS) is also considerably less than wild control, and active oxygen therein is clear
Except the enzymatic activity of agent (SOD, CAT, POD) is significantly higher than wild control.Result above all shows the American-European hybridization huge peak of grape variety
Overexpression of the adversity gene VlbZIP36 in arabidopsis improves the ability of the drought resisting stress of plant.
It is the specific embodiment that inventor provides below, to be further explained explanation to technical solution of the present invention.
The bioinformatic analysis of embodiment 1:VlbZIP36
The phyletic evolution of VlbZIP36 amino acid sequence and the bZIP amino acid sequence of other plant A and K groups studied
Tree analysis and its highly conserved amino acid residue Multiple sequence alignments the result shows that, VlbZIP36 amino acid sequence and arabidopsis
AtbZIP60 and rice Os bZIP74 amino acid sequence homology highest, and they belong to the K group subfamily in bZIP family
(Figure 1A).VlbZIP36 and other bZIP amino acid structures are analyzed, as a result, it has been found that all being wrapped in their amino acid sequence
The bZIP conserved domain (Figure 1B) being made of containing one DNA binding structural domain and leucine zipper motif.
The expression pattern analysis of embodiment 2:VlbZIP36
In order to analyze the expression pattern of VlbZIP36, inventor is analyzed comprising ProVlbZIP36: GUS expression vector turns base
Because of the GUS activity of arabidopsis.As a result, it has been found that cotyledon tip and root (Fig. 2 C) of the GUS in 5 -day-old of seedling, 2 week old seedling it is big
There is expression (Fig. 2 D) in portion of tissue, and without expression (Fig. 2A) in the mature embryo just sprouted.And after mannitol Stress treatment,
GUS has expressed (Fig. 2 B) at the tip for the mature embryo just sprouted, and the organized middle expression of institute is all aobvious in the seedling of 2 week old
Work property enhancing (Fig. 2 E).In adult seedling, GUS expression in the petiole (Fig. 2 F) of 3 week old, anther, style, silique (Fig. 2 L-O)
It is higher, there is fainter expression (Fig. 2 I) in guard cell, sepal, and without expression in petal.Drought stress processing and
After ABA processing, GUS is expressed in blade, petiole, guard cell enhances (Fig. 2 G, H, J, K).The result shows that VlbZIP36
Induction of the expression by arid and ABA in arabidopsis.
Resistance of the embodiment 3:VlbZIP36 transgenic arabidopsis strain to osmotic stress
To transgenic line and WT lines seed carry out osmotic stress processing the experimental results showed that, in MS culture medium
After upper culture 3 days, almost 100% transgenic line and wild type seeds can be sprouted, and in the mannitol containing various concentration
Culture medium on cultivate 3 days after, transgenic line then shows the seed germination rate (Fig. 3) of 21-28% higher than wild type.Simultaneously
After cultivating 6 days on osmotic medium, the cotyledon green percentage conspicuousness of transgenic line is higher than (Fig. 4 A, B) of wild type.Such as
Say, when mannitol concentration be 300mM when, all greenings of the cotyledon of about 83% transgenic line, and only 66% it is wild
The cotyledon greening of type plant；When mannitol concentration is 350mM, transgenic line then shows higher than wild type 27% or so
Survival rate.Inventor is analyzed again in different time sections later, and 350mM mannitol culture medium plants transgenic plant and wild type
The influence of strain survival rate.As shown in Figure 4 C, after osmotic stress is handled 3 days, the cotyledon of transgenic line and WT lines is equal
Non- greening, and after handling 7 days, the survival rate of transgenic line is then at least higher by 32% than wild type.
Plant can be enhanced to the resistance of osmotic stress to verify overexpression VlbZIP36, inventor analyzes transgenosis
Growing state of the arabidopsis strain seedling on the MS culture medium for being added to 300,350mM mannitol.Such as Fig. 5 A, shown in B, turn base
Because root system can be trained with normal growth in the MS of addition 300,350mM mannitol on MS culture medium for strain and WT lines
It supports in base, although the growth of transgenic line and WT lines root is suppressed, compared with the root system of WT lines,
The root system of transgenic line is more strong.
In order to further measure response of the VlbZIP36 transgenic line to osmotic stress, inventor determines Stress treatment
The relative conductivity and mda content of rear blade.The transgenic line that is grown in MS fluid nutrient medium and WT lines
Relative conductivity and mda content there are no significant difference, and after being grown in osmotic medium, the phase of transgenic line
(Fig. 5 C, D) lower than WT lines to conductivity and mda content shows the thin of under osmotic stress transgenic line
Born of the same parents' extent of damage is low compared with wild type.
In order to further verify function of the VlbZIP36 in terms of responding drought stress, inventor analyzes the quasi- south of transgenosis
Mustard strain is at age seedling by the situation after drought stress.As shown in fig. 5e, under normal growing conditions, the transgenosis of 3 week old
Strain and the growing way of WT lines are unanimous on the whole, and WT lines and transgenic line go out after drought stress processing 8 days
Existing blade is wilted and phenomenon of losing water, but symptoms are mild for the wilting of transgenic line.After rehydration 3 days, 53.13-65.63%'s turns
Gene strain is still also survived, and only 18.75% WT lines are survived.
Embodiment 4: overexpression VlbZIP36 can enhance Arabidopsis plant to the resistance of Water deficit
In order to further detect VlbZIP36 more details in terms of responding drought stress, inventor turns VlbZIP36
Gene strain and the whole strain plant of wild type are placed on clean filter paper, simulating drought experiment.As shown in Figure 6 C, entire dehydration
Cheng Zhong, transgenic line shows lower percentage of water loss compared with wild type.After dehydration 6 hours, with transgenic line phase
More serious blade is shown than WT lines here to wither (Fig. 6 A) and more cell death (Fig. 6 B).With morphological observation knot
Fruit is consistent, and wild-type leaves show higher relative conductivity (Fig. 6 D) and mda content (Fig. 6 E), shows scale
The dehydration resistance of arabidopsis can be enhanced up to VlbZIP36.
As fig 6 f illustrates, under the conditions of untreated, there was no significant difference for the stomatal aperture and wild type of transgenic line
(data are not shown), and after being handled blade 1 hour with 10 μM of ABA, the stomatal aperture of transgenic line and wild type all reduces
, but the former reduction is more, illustrates that transgenic line can enhance and is perhaps attributed to it to ABA's to the resistance of drought stress
Sensibility.
Embodiment 5:VlbZIP36 transgenic arabidopsis strain accumulates less active oxygen under drought stress, and accumulates more
More antioxidases
The method that inventor utilizes NBT and DAB histochemical stain has detected before dehydration respectively and plants after dehydration 6 hours
O in object blade cell2 -And H2O2Accumulating level.The result shows that before dehydration, WT lines and transgenic line blade
O2 -And H2O2Content does not have significant difference, and after dehydration 6 hours, the color that transgenic line is obviously showed than wild type
Shallower (Fig. 7 A, B), accumulated active oxygen is fewer in transgenic line compared with wild type for preliminary explanation, the oxidative stress received
It is smaller.Inventor determines the activity of SOD, CAT and POD before dehydration and in 6 hours rear blades of dehydration simultaneously.The result shows that
Before dehydration, the activity of SOD, CAT and POD do not have significant difference in wild type and transgenic line, and at dehydration 6 hours
Afterwards, compared with WT lines, SOD, CAT and POD activity in transgenic line are higher (Fig. 7 C-E).This result and dyeing
Observation is that consistent.
The expression of embodiment 6:qRT-PCR detection stress-related genes
In order to further appreciate that whether VlbZIP36 is involved in ABA signal path, inventor has detected dehydration by qRT-PCR
The expression quantity of the ABA of some known functions or stress-related genes in the wild type and transgenic line of 3 week old after processing.Knot
After fruit shows that dehydration is handled 1,2 hour, AtABI1, AtABI5, AtABF3, AtRD29A, AtRD29B and AtRAB18's is opposite
Expression quantity is all gradually increasing in wild type and transgenic line.However after dehydration 1 hour, in transgenic line
The expression quantity of AtABI5 gene has reached 2.6-3.1 times of wild control；The expression quantity of AtABF3 gene has reached wild control
1.2-1.5 times；The expression quantity of AtRD29A gene has reached 1.7-2.2 times of wild control；The expression quantity of AtRD29B gene
1.4-1.7 times of wild control is reached；The expression quantity of AtRAB18 gene has reached 1.6-2 times of wild control；And AtABI1
The expression quantity of gene is then slightly higher than wild control.After dehydration 2 hours, the expression quantity of AtABI1 gene reaches in transgenic line
1.8-2.4 times for having arrived wild control；The expression quantity of AtABI5 gene has reached 2.3-3 times of wild control；AtABF3 gene
Expression quantity reached 1.6-2 times of wild control；The expression quantity of AtRD29A gene has reached 1.7-2.4 times of wild control；
The expression quantity of AtRD29B gene has reached 1.4-2.1 times of wild control；The expression quantity of AtRAB18 gene has reached wild right
According to 1.3-1.5 times.It was unexpected that being normally used as ABA biosynthesis indicator in wild control and transgenic line
The expression quantity of AtNCED3 gene is risen after dehydration is handled 1 hour, and after dehydration is handled 2 hours, AtNCED3 base
The expression quantity of cause is declined again.Wherein, after dehydration 1 and 2 hour, the expression quantity of AtNCED3 gene in transgenic line
Respectively reached wild control 1.8-2.3 and 2.1-3.3 times.Meanwhile after dehydration is handled 1,2 hour, in ABA signal transduction mistake
Play the part of in journey AtERA1 the and AtKAT2 gene of negative regulating and controlling effect expression quantity be in wild type and transgenic line by
Gradually decline.After dehydration 1 and 2 hour, the expression quantity of AtERA1 gene has decreased to wild control respectively in transgenic line
0.56-0.63 and 0.5-0.6 times；The expression quantity of AtKAT2 gene have decreased to respectively wild control 0.76-0.83 and
0.65-0.7 times (Fig. 8).
Claims (1)
1. America and Europe's hybridization huge peak adversity gene VlbZIP36 of grape variety is used to improve the application of the drought resisting stress ability of arabidopsis,
The coding region sequence of the American-European hybridization huge peak adversity gene VlbZIP36 of grape variety is as shown in SEQ NO.1.
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| Title |
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| PREDICTED: bZIP transcription factor 60-like [Vitis vinifera];NCBI;《NCBI Reference Sequence: XP_003634336.1》;20111207;1 |
| PREDICTED: Vitis vinifera bZIP transcription factor 60-like (LOC100244512), mRNA;NCBI;《NCBI Reference Sequence: XM_003634288.1》;20111207;1 |
| 葡萄bZIP基因家庭的生物信息学及其表达分析;张宏静;《中国优秀硕士论文全文数据库（农业科技辑）》;20150315;6-42 |
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