CN105713909B - Wheat gene TaFUS3 and its application - Google Patents
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Abstract
The present invention relates to field of plant genetic project technology, in particular to a kind of wheat adversity gene TaFUS3 and its application.Wheat anti contravariance related gene TaFUS3 of the present invention is the separation from anti growing out kind " Huaihe River wheat 0360 " and easy Spike sprouting kind " Zhongyou9507 ", and full length gene cDNA is 1461 bp, the 5 '-noncoding regions including 47 bp；3 '-the noncoding regions of 484 bp；The code area of 906 bp, the poly-A tail of terminator and 28 bp, the albumen of the coded by said gene are the protein containing B3 structural domain that an overall length is made of 301 amino acid.Wheat anti contravariance related gene TaFUS3 of the invention can apply the degeneration-resistant reaction in seed development and suspend mode, anti growing out, breeding for stress tolerance, and genetic chip production designs the new anti contravariance related gene of synthesis and is of very high actual application value.
Description
Technical field
The invention belongs to field of plant genetic project technology, and in particular to a kind of wheat cdna TaFUS3 and its application.
Background technique
Plant transgene research starts from beginning of the eighties late 1970s, and nineteen eighty-three, Zambryski was obtained in the world
First case transgenic plant, Horsch in 1985 etc. create the leaf disk method in Agrobacterium-mediated Transformation, henceforth, establish respectively
Agrobacterium-mediated transformation, particle bombardment, electric shock perforation method, PEG mediated method, pollen tube passage method, microinjection, low energy ion beam
Mediated method etc..The successful species of transgenosis constantly expand, and are related to 35 section, 120 various plants, and wherein agrobacterium-mediated transformation obtains
Genetically modified plants account for 85% or more of transgenosis sum, and the genetically modified plants that particle bombardment obtains account for 10% or so.Dicotyledonous plant
Object is the natural host of Agrobacterium, and the gene transfer of a variety of dicotyledons is had been set up using Agrobacterium Ti plasmid mediated method
System, and some excellent foreign genes have been transferred to dicotyledon, it has been bred as transformed variety, but Agrobacterium is utilized to be situated between
The work progress that inducing defecation by enema and suppository carries out genetic transformation to monocotyledon is relatively slow.
Wheat is one of most important cereal crops, but the flow of research of its genetic engineering has lagged far behind other works
Object.Wheat transgenic research starts from the phase at the beginning of the eighties in last century.Vasil in 1992 etc. obtains generation using particle gun mediated method
First case wheat transgenic plant in boundary.1994 Nian Zengjun happinesses, Cheng Zhuomin etc. obtain wheat using pollen tube passage method respectively
Transgenic plant.GUS and npt II have been transferred to wheat using agrobacterium-mediated transformation for the first time by Cheng in 1997 etc., indicate agriculture bar
The breakthrough of bacterium mediated transformation wheat.Most, effect most preferably particle bombardment is applied in Efficiency of Wheat Transformation at present, absolutely mostly
Number transgenic wheat is obtained using this method.Application of the agrobacterium co-cultivation in transgenic wheat also reaches its maturity, and spends
Tube cell channel method is also taken seriously with its peculiar advantage.With the raising of Gene Isolation technology and cloning approach, will have from now on
More important gene related with wheat flour quality is cloned out, while some practical problems in Wheat Production, and such as high temperature is done
The problems such as non-irrigated, saline and alkaline, Spike sprouting and pest and disease damage, comes out in succession, and genetic engineering breeding may be best solution route.
Plant during the growth process, is sent out by the harm of various abiotic stress, including high temperature, arid, with high salt, fringe
Bud and pest and disease damage etc., high temperature and arid have highly important influence to the growth and development of wheat.According to statistics, every year by
The 20-25% of total cultivated area is accounted in the disaster area of the reasons such as high temperature, arid China wheat.Therefore, by the degeneration-resistant base of external source
Solution Wheat Production, a very important means of breeding are had become because importing wheat progress breed improvement.FUSCA3
(FUS3) be a kind of B3- type transcription factor, risen emphatically in seed development, seed dormancy and abiotic stress response
The regulating and controlling effect wanted.People isolate FUS3 gene from the plants such as arabidopsis, barley, long-grained nonglutinous rice, rape and in succession to its functions
Carried out Primary Study, and at present worldwide, in wheat about the separation of FUS3 gene, clone and anti growing out,
The research of degeneration-resistant reaction is also fewer.
Summary of the invention
The object of the present invention is to provide the applications of a kind of wheat cdna TaFUS3 and the gene.
The technical solution adopted by the present invention is as follows:
The full-length cDNA of wheat cdna TaFUS3 is 1465bp, the 5 '-noncoding regions including 47bp, the non-volume in the 3 '-of 484bp
Code area, the code area of 906bp, the poly-A tail of terminator and 28bp；Nucleotide sequence base composition are as follows: 387 a
358c 339g 381t, base sequence as: shown in SEQ ID NO:1.
The albumen of the described wheat cdna TaFUS3 coding is that an overall length by what 301 amino acid formed contains B3 structure
The protein in domain, amino acid sequence as: shown in SEQ ID NO:2.
Effect of the wheat cdna TaFUS3 in seed development and suspend mode.
Effect of the wheat cdna TaFUS3 in degeneration-resistant reaction.
Application of the wheat cdna TaFUS3 in anti growing out, breeding for stress tolerance.
Application of the wheat cdna TaFUS3 in genetic chip production.
Wheat cdna TaFUS3 synthesizes the application in new anti contravariance related gene in design.
Compared with prior art, this patent has following positive beneficial effect:
The present invention isolates wheat from anti growing out kind " Huaihe River wheat 0360 " and easy Spike sprouting kind " Zhongyou9507 "
TaFUS3 gene, and the sequence length, composition and alkali of the full-length cDNA of wheat cdna TaFUS3 are obtained by biological cloning technology
Basic sequence.
(1) wheat cdna TaFUS3 plays an important role in seed development and suspend mode.With the easy Spike sprouting kind of wheat " in it is excellent
9507 " seeds be research object discovery: wheat cdna TaFUS3 in Seed development, mainly after wheat is spent 25d embryo
In it is specific expressed, only have very micro expression in dry seeds embryo and young root；Wheat spends rear 35d (physiological maturity, suspend mode kind
Son) during Germination of Excised Embryos, the performance of TaFUS3 gene is in up-regulated expression trend；
(2) wheat cdna TaFUS3 plays an important role in degeneration-resistant reaction.Wheat cdna TaFUS3 is an environment stress
Response gene, when respectively by with high salt, ABA, mannitol, high temperature stress, expression is significantly increased；
(3) application of the wheat cdna TaFUS3 in anti growing out, breeding for stress tolerance: the gene is inserted into different type
Promoter downstream, be built into plant expression vector, pass through technique for gene engineering carry out plant stress-resistance improvement；
(4) application of the wheat cdna TaFUS3 in genetic chip production: with the cDNA of the wheat cdna TaFUS3 of clone
For template, DNA product is obtained by PCR amplification with gene-specific primer, after being purified, being dissolved to product, according to gene
Chip manufacturing program making wheat cdna chip is used for the correlative studys such as anti growing out, degeneration-resistant；
(5) wheat cdna TaFUS3 synthesizes the application of new adversity gene in design: according to TaFUS3 gene and HvFUS3
HvFUS3 gene is connected with TaFUS3 gene, synthesizes completely new gene by the difference of gene.Then, it is closed by sequence verification
It is whether correct at the encoder block (ORF) of gene, and will be new gene constructed into plant expression vector, pass through transgenic technology pair
The resistance of synthesis gene is detected, and the synthesis gene with good resistance can be used for the degeneration-resistant new variety of wheat of transgenosis
Cultivation.
Detailed description of the invention
Expression of Fig. 1 TaFUS3 gene during Wheat Development, in figure, abscissa is indicated: during Wheat Development
Tissue；Ordinate indicates: the relative expression quantity of wheat TaFUS3 gene；
Expression of Fig. 2 TaFUS3 during 35d Germination of Excised Embryos after wheat is spent, in figure, abscissa is indicated: after wheat is spent
The processing time of 35d In vitro Embryo, ordinate indicate: the relative expression quantity of wheat TaFUS3 gene；
Degeneration-resistant expression of Fig. 3 wheat cdna TaFUS3 under different condition of culture, in Fig. 3 (A), abscissa is indicated: wheat
Time of the seed in culture dish (A), ordinate indicate: the relative expression quantity of wheat TaFUS3 gene；In Fig. 3 (B), abscissa
Indicate: time of the wheat seed in culture dish (B), ordinate indicate: the relative expression quantity of wheat TaFUS3 gene；Fig. 3 (C)
In, abscissa indicates: time of the wheat seed in culture dish (C), and ordinate indicates: the relative expression of wheat TaFUS3 gene
Amount；In Fig. 3 (D), abscissa is indicated: time of the wheat seed in culture dish (D), ordinate indicate: wheat TaFUS3 gene
Relative expression quantity.
Specific embodiment
It is with the following Examples and attached drawing, right in order to keep the objectives, technical solutions, and advantages of the present invention more clear
The present invention is described in further detail, but protection scope of the present invention is not limited to following embodiments.
Embodiment 1: the cloning procedure of wheat cdna TaFUS3
(1) wheat cdna TaFUS3 is from anti growing out kind " Huaihe River wheat 0360 " and easy Spike sprouting kind " Zhongyou9507 "
Separation.With above-mentioned two kind, 21,28 and 35 days seeds of Hua Hou are material extraction total serum IgE, further separate mRNA, lead to
It crosses reverse transcription and mRNA is transcribed into cDNA, be that template carries out gene cloning with the cDNA.
(2) a pair of of degenerate primer is devised according to the plant FUS3 gene conserved functional domains that GenBank is logged in (to draw upstream
Object P1:5 '-WGGGAGAATTGTTCTYCCRAA-3 ' and downstream primer P2:5 '-ARTACCTSACRTTSGCTACYG-3 '), lead to
It crosses reverse transcription PCR technology and obtains the cDNA segment that a molecular weight is about 470bp, amplified production is recovered to be cloned in pMD18-
It is a part of TaFUS3 gene by sequence analytical proof this cDNA segment on T Eseay carrier, after sequencing.In gained
On the basis of cDNA segment, and two nest-type PRC primer 5 ' GSP1 (sequences are as follows: 5 '-are devised at the cDNA segment 5 ' end
GCAACTAGATCATCTCCCGCCTTCT-3 '), 5 ' GSP2 (sequence are as follows: 5 '-GAAGTCTCCCACTCGAAGGTTATGG-3 '),
3 ' ends devise a 3 ' GSP of gene-specific primer (sequence are as follows: 5 '-AGAAGGCGGGAGATGATCTAGTTGCT-3 ').It is logical
It crosses 5 ' and 3 ' Rapid amplification of cDNA ends (5 ' and 3 '-RACE) and obtains full length cDNA clone, by the full-length cDNA of acquisition
It is inserted on pMD18-TEseay carrier, and is stored in e.colistraindh5α.
Specific cloning procedure:
The synthesis of cDNA: total serum IgE is extracted with TRIzol reagent.It is isolated with Poly (A) Tract mRNA separation system Ш
mRNA.Use SMARTTMRACE cDNA amplification kit synthesizes cDNA.
Reverse transcription PCR: using cDNA as template, and degenerate primer P1 and P2 is designed and carries out PCR amplification.PCR reaction system is
20 μ L: contain 10 × PCR Buffer, 2.0 μ L, 10mmolL-1dNTP 1.2μL、10μmol·L-1Each 1 μ L of primer, 5U μ L- 10.2 μ L of Taq polymerase, 1 cDNA μ L, use ddH2O polishing is to 20 μ L.PCR amplification program are as follows: first 95 DEG C initial denaturation 3 minutes；35
95 DEG C of a circulation are denaturalized 30 seconds, and 53 DEG C are annealed 30 seconds, and 72 DEG C extend 1 minute；Last 72 DEG C extend 10 minutes.
Rapid amplification of cDNA ends: special kit (SMART is usedTMRACE cDNA Amplification Kit)
It is expanded.Solution is prepared by specification and is carried out, and 5 '-RACE amplification programs are divided into two steps: step 1: using 5 ' GSP1 and
SMARTTM5 ' cap the nested primers of RACE cDNA Amplification Kit, are expanded using touchdown PCR, and PCR expands
Increase program be 95 DEG C initial denaturation 4 minutes；95 DEG C of 20 circulations are denaturalized 30 seconds, and 62 DEG C are annealed 30 seconds, and 72 DEG C extend 3 minutes；10
95 DEG C of a circulation are denaturalized 30 seconds, and 58 DEG C are annealed 30 seconds, and 72 DEG C extend 3 minutes；Finally extend 10 minutes at 72 DEG C；Step 2:
Carry out PCR amplification using 5 ' GSP2 and 5 ' cap nested primer of kit, PCR reaction condition be 95 DEG C initial denaturation 4 minutes；36
95 DEG C of circulation are denaturalized 30 seconds, and 54 DEG C are annealed 30 seconds, and 72 DEG C extend 3 minutes；Last 72 DEG C extend 10 minutes.3 '-RACE amplification
Program are as follows: apply 3 ' GSP and SMARTTM3 ' the special primers of RACE cDNA Amplification Kit carry out PCR amplification,
Amplification program is identical as 5 '-RACE first step PCR amplification programs.
Product recycling and sequencing analysis: amplified production is separated by electrophoresis on 1% Ago-Gel.Target DNA will be contained
The Ago-Gel of segment is cut down, and recycles target DNA fragment with Ago-Gel QIAquick Gel Extraction Kit.The DNA fragmentation of recycling
After being dissolved with ultrapure water, it is connect with pMD18-T Eseay carrier with T4-DNA ligase, 4 DEG C of connections are overnight.Use CaCl2
Thermal shock method, 42 DEG C thermal shock 90 seconds, connection product is transformed into e.colistraindh5α.It is extracted with small amount plasmid extraction method
Plasmid DNA.In Beijing, Hua Da gene Co., Ltd is sequenced.Sequencing result BLASt and BLAStp computer aided program
(Altschul etc. 1997) American National Bioinformatics Institute website NCBI (http://www.ncbi.nlm.nih.gov) and
The web site of associated carries out homologous sequence inquiry and compares.The website NCBI with the method for Marchler-Bauer etc. (2003) into
The analysis of row conserved functional domains data.
(3) similarity query comparison is carried out in GenBank, it was demonstrated that the full-length cDNA cloned is new wheat cdna,
It is named as TaFUS3.Shown by the conservative domain analysis of NCBI, which belongs to B3 superfamily, sequence with cloned
Barley HvFUS3 gene (Moreno-Risueno etc. 2008；GanBank query ID:AM418838) highly similar.It compares
As a result as follows:
Above comparison result can illustrate wheat cdna TaFUS3's and barley of the invention from molecular structure
HvFUS3 gene has similar effect, it may be assumed that important regulating and controlling effect is played in seed development and suspend mode.
Embodiment 2: effect of the wheat TaFUS3 gene in seed development and suspend mode
Effect of the wheat cdna TaFUS3 in seed development: it is mature full to choose the easy Spike sprouting kind " Zhongyou9507 " of wheat
Full consistent seed uniform in size, sterilized be placed in culture dish are germinateed, and germination 10d, which is moved back in soil, to be grown, and are entirely being given birth to
The root and leaf, young fringe, Hua Hou of 10d after the primary root of 1d and bud, germination after the embryo of mature seed, germination are taken in long period respectively
The material as gene development spatial and temporal expression characteristic: the embryo and endosperm of the seed of 25d, Hua Hou 25d use real time fluorescent quantitative
PCR analytic approach, analyze wheat TaFUS3 gene relative expression quantity, result as shown in Figure 1: TaFUS3 gene mainly in wheat
Spend specific expressed in the embryo of rear 25d, the only very micro expression in dry seeds embryo and young root.
Effect of the wheat cdna TaFUS3 in seed dormancy: the easy Spike sprouting kind " Zhongyou9507 " of wheat and anti-fringe is taken to send out
Bud kind " Huaihe River wheat 0360 " spends rear 35d mature and plump consistent seed uniform in size, with 70% alcohol disinfecting 1min, sterile water
It rinses 3 times, with 0.1% HgCL26min is sterilized, after aseptic water washing 4~5 times, removes kind of a skin with dissecting needle and strips embryo, will shell
Under embryonic shield piece be placed on MS solid medium downward, 3 repetitions, 25 DEG C of dark cultures are set.Culture 0d, 2d, 3d are taken respectively
Embryo afterwards analyzes wheat using real-time fluorescence quantitative PCR analytic approach as the material of gene suspend mode spatial and temporal expression characteristic
The relative expression quantity of TaFUS3 gene, result are as shown in Figure 2: (1) 35d (physiological maturity) Germination of Excised Embryos after wheat is spent
In the process, the performance of TaFUS3 gene is in up-regulated expression trend.Compared with untreated 0d, 2d after treatment, TaFUS3 gene
Modulation is up to 5 times or more in expression；(2) 2d after treatment, TaFUS3 gene are in anti growing out kind " Huaihe River wheat 0360 "
Middle expression quantity is apparently higher than easy Spike sprouting kind " Zhongyou9507 ", and significant difference.
Embodiment 2 has been experimentally confirmed TaFUS3 gene and has played important regulating and controlling effect in seed development and suspend mode.
Quantitative fluorescent PCR analytic approach specific steps are as follows: referring to TaKaRa SYBR Green qPCR kit
The real-time fluorescence quantitative PCR primer principle of specification designs TaFUS3 gene qRT-PCR primer P5:5 '-
GTGGCTGGGTTGCGAGTTAT-3 ' and P6:5 '-ATCCTGCTCTTGTTGTTTGGC-3 ', logged according to GenBank (GB:
AB181991 wheat house-keeping gene (β-Actin) cDNA sequence) designs special primer P7:5 '-
TTTGAAGAGTCGGTGAAGGG-3 ' and P8:5 '-TTTCATACAGCAGGCAAGCA-3 ', real-time fluorescence quantitative PCR reactant
System is 20 μ L: Master containing qPCR Mix (2 ×), 10 μ L, 2 μ L of cDNA template, 10 μm of olL-1Each 1.0 μ L of forward and reverse primer
With 6.0 μ L of Nuclease-Free Water, each sample is arranged 3 repetitions, reacts in Bio-Rad CFX ConnectTM
It is carried out on Real-Time System.(reaction condition be 95 DEG C initial denaturation 30 seconds, 40 circulation 95 DEG C be denaturalized 15 seconds, 60 DEG C
Annealing 30 seconds, 72 DEG C extend 30 seconds, collect fluorescence signal in 60 DEG C of reaction steps, determine each gene phase according to amplification curve
The Ct value answered, the copy number of pcr template is corrected using β-actin as internal reference, and relative quantity uses 2-ΔΔCtMethod calculates).
Embodiment 3: effect of the wheat TaFUS3 gene in degeneration-resistant reaction
Selection wheat breed " Zhongyou9507 " mature and plump consistent seed uniform in size, 70% alcohol disinfecting 1 minute,
Aseptic water washing 3 times, with 0.1% HgCL2After disinfection 6 minutes, aseptic water washing 4-5 times, four parts are divided into, is respectively placed in
It is placed with the middle progress Stress treatment of culture dish (A), (B), (C), (D) of two layers of sterilizing filter paper.Wherein, 8mL is added in culture dish (A)
NaCl (150mmoL/L) solution, culture dish (B) is middle to be added 8mL abscisic acid (ABA, 30 μm of oL/L) solution, adds in culture dish (C)
Enter 8mL mannitol (300mmoL/L) solution, (the natural lighting time is 12 hours/day, and weather room temperature is in phjytotron
20 DEG C) culture.Be added in culture dish (D) 8mL aqua sterilisa be placed in 32 DEG C of illumination boxs (light application time and weather room temperature with
Other stress conditions are identical) in carry out high temperature stress processing, above-mentioned processing is respectively provided with 3 repetitions.Using real-time fluorescence quantitative PCR
Analytic approach, 0,6,12,24,48 hour imbibition seed is as wheat after taking culture dish (A), (B), (C), (D) middle processing respectively
The material of TaTUS3 adverse-resistant characteristic analyzes the relative expression quantity of wheat TaFUS3 gene, and result is as shown in Figure 3: TaFUS3 base
Because when respectively by with high salt, ABA, mannitol, high temperature stress, expression is significantly increased.Compared with coercing 0h, after coercing 36h,
TaFUS3 gene expression enhances amplitude respectively up to 36,56,55 and 35 times (Fig. 3).The above results explanation, TaFUS3 gene is one
A Stress response gene is expressed obvious when by environment stress.
Embodiment 4: application of the wheat cdna TaFUS3 in anti growing out, breeding for stress tolerance
TaFUS3 gene high-fidelity Taq-DNA polymerase and gene-specific primer are expanded, connected with T4-DNA
The downstream that TaFUS3 is connected to specifically-expressed in rice embryo promoter ESG (Embryo Specific_Gene) by enzyme is connect, then is inserted
Enter into pFGC5941-Bar plant expression vector, to be built into pFGC5941-ESG-FUS3 expression vector, imports non-anti- fringe
In wheat breed, anti growing out breeding is carried out；Or the gene is connected to maize ubiquitin efficient promoter Ubi
(Ubiquitin-1) downstream is inserted into pFGC5941-Bar plant expression vector, to be built into pFGC5941-
Ubi-FUS3 expression vector plasmid imports in the poor wheat breed of resistance, carries out wheat breeding for stress tolerance.Above-mentioned expression is carried
Constitution grain, which is transformed into e.colistraindh5α, breeds, and extracts the plasmid of the carrier, will by Agrobacterium-mediated Transformation method
PFGC5941-ESG-FUS3 or pFGC5941-Ubi-FUS3 expression vector plasmid is transformed into high yield, high-quality and poor resistance small
In the stem-tip tissue of wheat variety, transgenosis breeding for stress tolerance can be carried out.
The concrete operation step of Agrobacterium-mediated Transformation method transgenosis breeding for stress tolerance are as follows: (1), by wheat seed disinfect postposition
In the culture dish added with filter paper, 2~3d is sprouted under 25 DEG C of illumination conditions；(2), by pFGC5941-Ubi-FUS3 expression vector
Plasmid imports in Agrobacterium tumefaciems EHA105, and picking positive monoclonal is inoculated into added with 50mg/L kanamycins and 50mg/L
In the liquid YEP medium of rifampin, 28 DEG C, 160rpm is incubated overnight；(3), by the EHA105 bacterium after step (2) are cultivated
Liquid, 2000rpm abandon supernatant after being centrifuged 1min, and sediment is hanged again with the YEP culture medium added with 100 μm of ol/L acetosyringones
Floating culture, 28 DEG C, 160rpm, until absorbance reaches 1.0 or so；(4), when the wheat malt in step (1) it is long to 1~2cm when,
Bud directly is got rid of, exposes basal growth cone, and the seed for getting rid of bud is immersed directly in EHA105 obtained in step (3) and is hanged
30s is rocked in supernatant liquid, infects seed sufficiently；(5), the wheat seed disseminated is transferred to the new culture added with filter paper
In ware, 25 DEG C of dark culture 2d, 25 DEG C illumination cultivation one week or so, move to flowerpot to the three pieces young leaves period of the day from 11 p.m. to 1 a.m wait grow two, until at
It is ripe, transgenic wheat is obtained in this way；(6), using on expression vector riddled basins-herbicide-resistant gene Bar and
Gene-specific primer is detected by offspring of the round pcr to transgenic plant, is selected gene pure, anti growing out, is resisted
The transgenic strain that inverse performance significantly improves can carry out field planting, promote.
Embodiment 5: application of the wheat cdna TaFUS3 in genetic chip production
Using the cDNA clone of TaFUS3 gene as template, DNA product is obtained by PCR amplification with gene-specific primer,
After being purified, being dissolved to product, wheat cdna chip is made according to genetic chip production process, for anti growing out, degeneration-resistant
Equal correlative studys.Using the nylon membrane that the automatic point film instrument of the BioRobotics of Britain is positively charged in 8 × 12cm by specimens point
On, 2 points of each sample spot, the amount of DNA of each point is 5 nanograms, with house-keeping gene on time point (β-actin gene) as stabilization
Expressing gene control after experimental study material (including control group and experimental group) is extracted total serum IgE, carries out reverse transcription generation respectively
Then control group and the cDNA of experimental group are marked Cy3 and Cy5 fluorescence signal as probe by cDNA respectively, with genetic chip into
Row hybridization.Using laser scanner scans chip analysis hybridization signal, according to the strong and weak analysis different experimental conditions of hybridization signal
The expression of lower TaFUS3 gene changes, the relationship that research TaFUS3 gene is reacted with anti growing out, degeneration-resistant etc..
Embodiment 6: wheat cdna TaFUS3 synthesizes the application in new anti contravariance related gene in design
According to the difference of TaFUS3 gene and HvFUS3 gene, it is pervious that HvFUS3 gene is encoded into 60 amino acids residues
77 amino acids residues are encoded with TaFUS3 gene after cDNA synthesis to be connected to gene cDNA end section, are thus synthesized
Completely new gene.Then, whether the encoder block (ORF) for synthesizing gene by sequence verification is correct.Pass through the gene of verifying
It is further building up in plant expression vector, is detected, had good by resistance of the transgenic technology to synthesis gene
The synthesis gene of resistance can be used for the cultivation of the degeneration-resistant new variety of wheat of transgenosis, the building of plant expression vector and turn base
The concrete operations of cause are identical with described in embodiment (4).
Claims (5)
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| CN104910263A (en) * | 2014-03-12 | 2015-09-16 | 中国农业科学院作物科学研究所 | Plant stress tolerance associated protein TaPPR, and encoding gene and application thereof |
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| CN104910263A (en) * | 2014-03-12 | 2015-09-16 | 中国农业科学院作物科学研究所 | Plant stress tolerance associated protein TaPPR, and encoding gene and application thereof |
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