CN1762201A - A kind of method that strengthens drought resistance of wheat - Google Patents
A kind of method that strengthens drought resistance of wheat Download PDFInfo
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- CN1762201A CN1762201A CN 200510104870 CN200510104870A CN1762201A CN 1762201 A CN1762201 A CN 1762201A CN 200510104870 CN200510104870 CN 200510104870 CN 200510104870 A CN200510104870 A CN 200510104870A CN 1762201 A CN1762201 A CN 1762201A
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Abstract
The invention discloses a kind of method that strengthens drought resistance of wheat.The method of enhancing drought resistance of wheat provided by the present invention, it is encoding gene insertion plant expression vector with the DREB transcription factor, obtain containing the recombinant expression carrier of the encoding gene of DREB transcription factor, this recombinant expression carrier is imported in wheat, obtain the wheat that drought resistance strengthens.Method of the present invention can significantly improve the drought resistance of wheat.
Description
Technical field
The present invention relates to a kind of method that strengthens drought resistance of wheat.
Background technology
Arid is one of main environment limiting factor that has a strong impact on plant growth and output, because the characteristic that is difficult to predict, the seed selection of drought-resistant variety is more much more difficult than the seed selection of other resistant variety.Wheat is the same with other crop, is easily in theory though control drought resistance from the angle of gene, and is difficult in the breeding work of reality.Although traditional drought resisting breeding method has obtained certain achievement in the past, a large amount of consumption time and manpower, and obtained effect is not very remarkable in the arid producing region of whole world wheat.
Develop rapidly along with molecular biological, utilize the engineered method of drought-resistant plant to increase the resistance of wheat, thereby the molecular breeding mode that improves wheat yield is progressively becoming the main body of breeding work from now on arid.
Under drought stress, the gene of expression of plants is divided into two classes: 1. the product of gene code is some functional proteins and enzyme, as LEA, and aquaporin, proline synzyme etc.; 2. the product of gene code is a transcription factor of transmitting signal and regulate gene expression, as bZIP, and MYC, DREB etc.Because the product of first kind gene is clear and definite, research more thorough obtained some drought-enduring genetically modified plants at present.For second genoid,, become the emphasis of anti-drought gene engineering at present in view of these expression of gene are regulated and control a series of functional genes in its downstream.DREB (Dehydration Responsive Element Binding) albumen (transcription factor) is a cis-regulating element of finding in the mouseearcress, the DREB transcription factor belongs to the EREBP type transcription factor in the AP2/EREBP family that has found at present, comprises many members such as DREB1A～C and DREB2A～B.Amino acid sequence analysis shows that the DREB transcription factor all contains the acid transcriptional activation domain of C-end, the terminal alkaline nuclear localization signal of N-.Their AP2/EREBP domain is made up of 58 amino acid residues, contains the conserved sequence that can form 3 beta sheets and 1 alpha-helix, and both can both be the DRE combination of elements of PuCCGAC with core sequence.DREB1A and the regulation and control and low temperature and the relevant rd17 of arid high-salt stress patience respectively of DREB2A transcription factor in the mouseearcress; kin1; cor6.6; cor15a, several genes such as erd10 and rd29A, not only said gene is expressed under home; under arid or low temperature treatment condition; their expression also significantly strengthens than wild type, and can cause the raising of proline content, thereby the many-sided resistance of plant (drought resisting, freeze proof and anti-salt) is improved.
The Drought-resistance in Wheat molecular breeding mode that biotechnology approach and gene engineering make anti-drought gene change the acceptor plant over to is achieved, but can anti-drought gene successfully change over to, can genes of interest express, can the acceptor plant show the proterties that foreign gene is controlled, and also needs to identify by a series of experiment.In wheat, drought resistance of wheat has seedling stage, and the branch of later stage and drought resisting in the time of infertility carries out organ and build up seedling stage, and establishes material base for output.Seedling stage, drought resistance became the necessary ways of first screening.
Summary of the invention
The purpose of this invention is to provide a kind of method that strengthens drought resistance of wheat.
The method of enhancing drought resistance of wheat provided by the present invention, it is encoding gene insertion plant expression vector with the DREB transcription factor, obtain containing the recombinant expression carrier of the encoding gene of DREB transcription factor, this recombinant expression carrier is imported in wheat, obtain the wheat that drought resistance strengthens.
Described plant expression vector can be Ti class plasmid vector or viral vectors or the conventional carrier of particle gun conversion.
The encoding gene of described DREB transcription factor can derive from mouseearcress or wheat or paddy rice or corn or soybean or rape etc.
The encoding gene of described DREB transcription factor is preferably and derives from mouseearcress.
The encoding gene of described DREB transcription factor is the dna fragmentation from 5 ' end the 612nd to 1320 bit base with Genbank Access No.AB013816.For the ease of inserting expression vector, can add the BamHI site in 5 ' design, add the SacI site in the design of 3 ' end, sequence total length 727bp, as the sequence in the sequence table 1, from the 1st to the 6th deoxynucleotide of 5 ' end is the BamHI recognition site, is the SacI recognition site from the 722nd to the 727th deoxynucleotide of 5 ' end.
In described recombinant plasmid expression vector, start promotor that the encoding gene of described DREB transcription factor transcribes and be adverse circumstance induction types such as the promotor of the hydrophilic albumen RD29A of promotor, mouseearcress gene of the hydrophilic albumen RD29B of mouseearcress gene or SAG12 promotor; Or constitutive promoters such as corn polyubiqutin gene promoter, cauliflower mosaic virus CaMV 35S.
The promotor of the hydrophilic albumen RD29B of described mouseearcress gene, contain Genbank Access No.D013044 from 5 ' dna fragmentation of end the 85th to 1784 bit base.For the ease of inserting expression vector, can add the HindIII site in 5 ' design, add the BamHI site in the design of 3 ' end, sequence total length 1706bp, as the sequence in the sequence table 2, from the 1st to the 6th deoxynucleotide of 5 ' end is the HindIII recognition site, is the BamHI recognition site from the 1701st to the 1706th deoxynucleotide of 5 ' end.
The recombinant expression carrier of the encoding gene of the DREB transcription factor of the described RD29B of containing promoters driven is pBAC123 (5102bp); Or be to insert bar gene and the NOS terminator sequence that the CaMV 35S promoter drives in the HindIII site of pBAC123, obtain with the recombinant expression carrier pBAC128F (7024bp) of bar gene as selected marker.
In the described method, be to be explant with wheat children tassel or rataria, the recombinant expression carrier that will contain the encoding gene of DREB transcription factor imports wheat.
The recombinant expression carrier that carries the encoding gene of DREB transcription factor can be by particle bombardment, use that Ti-plasmids, Ri plasmid, plant viral vector, microinjection, electricity be led, conventional biological method transformed wheat cell or tissue such as agriculture bacillus mediated.
The described recombinant expression carrier that carries the encoding gene of DREB transcription factor preferably imports wheat by particle bombardment.
Described particle gun transformation system, with nonselective herbicide PPT (Phosphinothricin, trade name Basta), or bialaphos, or Glufosinate ammonium (careless ammonia phosphine, Sigma) selective agent of conduct conversion resistance.The present invention adopts PPT as selective agent.
The method of enhancing drought resistance of wheat of the present invention is particularly suitable for following wheat breed: 8901, and 5-98,99-92 and Bao Feng 104.
The present invention is with winter wheat variety 8901,5-98, the young fringe and the rataria of 99-92 and kinds such as 104 are material, transform with particle gun to contain adverse circumstance induced transcription factor DREB (by the promoters driven of the hydrophilic albumen RD29B of mouseearcress gene) and the bar gene recombinant expression carrier pBAC128F (7024bp) as selected marker; Through screening and plant regeneration, obtain more than 70 transgenic wheat plant and offspring's strain thereof system altogether.Transgenic line is through physical signs analyses such as pcr analysis, the detection of RNA dot blot and plant proline contents, the result show external source transcription factor DREB gene stable integration in transfer-gen plant and offspring's strain thereof system, and in part offspring strain system, obtained expression.The blade proline content is measured and to be shown, has the proline content of 16 transgenic lines to compare with the non-transgenic contrast, increases quite significantly, and wherein the proline content of 10 strain systems is more than 1100 μ g/g, and comparison is shone and improved more than 2 times.Indoor drought resisting constructed experiment shows that after transgenic line stopped to water 15 days, blade still showed green, and the contrast blade is chlorosis, dried-up then; After the rehydration 10 days, transgenic line rejuvenates, and contrast is then dead.Experimental result shows, utilizes adverse circumstance inducible promoter (RD29B) to strengthen the expression of external source dreb gene, can significantly improve the drought resistance of wheat.
Description of drawings
Fig. 1 is the structural representation of plant expression vector pBAC128F
Fig. 2 is T
0PCR for transfer-gen plant detects
Fig. 3 is T
1Dot blot for transfer-gen plant is identified
Fig. 4 is T
1PCR for transfer-gen plant detects
Fig. 5 is non-transgenic plant and 7 transfer-gen plant T
1The total RNA electrophoresis pattern of blade for strain system
Fig. 6 is the proline content that arid is handled five days rear section transgenic wheats
Fig. 7 A is the growing state of non-transgenic plant after stopping to water 15 days
Fig. 7 B stops to water the growing state of 15 days rear section transfer-gen plants
Fig. 7 C is the growing state of rehydration non-transgenic plant after 10 days
Fig. 7 D is the growing state of 10 days rear section transfer-gen plants of rehydration
Embodiment
Experimental technique among the following embodiment if no special instructions, is conventional method.
T
0The transfer-gen plant that expression is obtained by the callus of wheat, T
1Expression T
0The seed that produces for selfing and by plant that it grew up to.
1, the structure of plant expression vector pBAC128F
The physical map of plant expression vector pBAC128F as shown in Figure 1, concrete construction method is as follows:
(1) clone of dreb gene
Dreb gene sequence (GenbankAccess No.AB013816) according to the mouseearcress of having delivered (Arabidopsis thaliana), design and synthesize the special primer of this gene: P1: upstream primer 5 '-GGATCCTGATCAATGAACTCATTTTC, downstream primer P2:5 '-GAGCTCCCATTCTAAAAAAGGAAC, genomic DNA with mouseearcress is a template, utilizes PCR to clone this gene.Wherein, the temperature condition of PCR is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 45s then, 45 ℃ of annealing 45s, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 10min.About 730bp dna fragmentation that amplification is obtained reclaims this fragment with the scheme that gel reclaims kit (sky, Beijing is a Time Technology Co., Ltd) and provides by supplier, with (Promega company of pGEM-Teasyvector system, the U.S.) scheme that provides by supplier is cloned this fragment, order-checking (Shanghai Shen You biotech company), sequencing result shows that this fragment has the nucleotide sequence of sequence 1 in the sequence table.By with dreb gene sequence (Genbank Access No.AB013816) relatively, confirm to be cloned into have Genbank Access No.AB013816 from the 632nd to 1320 gene order fragment of 5 ' end.
(2) structure of dreb gene expression vector pBAC128F
The construction method of plasmid pBAC128F, process and genetic transformation are with reference to " molecular cloning " second edition (SambrookJ, Fritsch E F, Maniatis T.Molecular Cloning-A Laboratory Manual.2nded.New York:Cold Spring Harbor laboratory Press, 1989).Concrete grammar is as follows:
1) with SacI and EcoRI double digestion plasmid pSP72 (available from Promega company), Agrobacterium tumefaciems rouge alkali synthetase gene NOS 3 ' the terminator sequence of inserting about 260bp is (from plasmid vector pBARGUS, with SacI and EcoRI double digestion, Vasil, et al, Bio/technology, 10:667-674,1992).The plasmid that is built into is named and is pBPC18.
2) the about 730bp dreb gene fragment that is obtained by the PCR clone adopts the TA cloning process to insert pGEM-T (Promega), after the dna sequencing checking is correct, and with BamHI and SacI double digestion, the BamHI and the SacI site of inserting pBPC18.The plasmid that is built into is named and is pBAC118.
3) genomic DNA by mouseearcress is a template, upstream primer P1:5 '-GCGGAAGCTTCATTTTCTGCTACAG, downstream primer P2:5 '-GGATCCTTTCCAAAGCTGTGTTTTCTCTTTTTC carries out the 1.7kb RD29B promoter fragment (dna fragmentation from 5 ' end the 85th to 1784 bit base of Genbank Access No.D013044) that the PCR clone obtains, adopt the TA cloning process to insert pGEM-T (Promega), after the dna sequencing checking is correct, with HindIII and BamHI double digestion, insert between the HindIII and BamHI site of pBAC118.The plasmid that is built into is named and is pBAC123.
4) plasmid pBAC123 cuts with the HindIII enzyme, the CaMV 35S promoter that inserts two ends and be the HindIII cohesive end is fragment (Vasil, et al, the Bio/technology of terminator with the bar gene of corn Adh1 intron driving and with NOS 3 ', 10:667-674,1992).The plasmid forward that makes up inserts and names into pBAC128F (Fig. 1), oppositely inserts to name to be pBAC128R.
The genes of interest DREB (DREB11B) of pBAC128F is driving with the promotor (1.7kb) of the hydrophilic albumen RD29B of mouseearcress gene, is adverse circumstance abduction delivering type.Contain bar gene that the Intron1 of CaMV 35S promoter and corn Adh1 gene drives simultaneously as selected marker.
2, plant expression vector pBAC128F transformed wheat
With winter wheat 8901,5-98,99-92 and Bao Feng 104 kinds are the acceptor kind, choose young fringe, rataria is an explant, according to document (Zhang Xiaodong, Liang Rongqi, Chen Xuqing, Deng. the acquisition of high-quality HMW gluten subunit transgenic wheat and genetic stability thereof and quality trait analysis. Science Bulletin, 2003,48 (5): the 474-479) method of Miao Shuing transforms explant with pBAC128F with the particle gun micropellet bombardment, the explant of conversion is selected on the medium that contains weed killer herbicide PPT, and and then through differentiation regeneration cultivation acquisition 72 strain T
0For transgenic regenerated plant, wherein protect rich 104 (13 strains), 8901 (31 strains), 5-98 (26 strain), 99-92 (2 strain).
3, the analysis of molecules of transfer-gen plant
(1) PCR of transfer-gen plant detects and the dot blot analysis
(i) T
0PCR for transfer-gen plant detects
Extracting genome DNA adopts the CTAB method.The PCR of transfer-gen plant detects, and uses dreb gene upstream primer P1 and downstream primer P2, with the negative contrast of unconverted plant, and the positive contrast of plasmid pBAC128F.Reaction system is: cumulative volume is 25 μ l, 10 * PCR buffer solution, 2.5 μ l, each 1 μ l of primer (10 μ M), dNTPs 2 μ l, ddH
2O 13 μ l, Taq enzyme 1U, MgCl
22 μ l, template DNA 1 μ g.Its cyclic amplification program is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 45s then, 45 ℃ of annealing 45s, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 10min.The PCR testing result shows that having 4 strains to increase in 13 strains, the 104 kind transfer-gen plants obtains the specific fragment of the dreb gene about 724bp, 31 strains, 8901 kind transfer-gen plants have 6 strains to increase to obtain the specific fragment of the dreb gene about 724bp, there are 16 strains to increase in the 26 strain 5-98 kind transfer-gen plants and obtain the specific fragment of the dreb gene about 724bp, have 1 strain to increase in the 2 strain 99-92 kind transfer-gen plants and obtain the specific fragment of the dreb gene about 724bp.T
0For the PCR testing result of transfer-gen plant as shown in Figure 2, show the part transfer-gen plant (swimming lane 5 that is obtained, 6,9,10 etc.) can both amplify the specific fragment of the dreb gene about 724bp with positive control plasmid (swimming lane 1), negative control (no template DNA, swimming lane 2) and non-transgenic plant (swimming lane 3) do not have amplified band, show that the external source dreb gene has entered transfer-gen plant.Among Fig. 2, swimming lane 1 plasmid pBAC128F; Swimming lane 2 negative controls (water); Swimming lane 3 non-transgenic plant; Swimming lane 4-19 shows 104 respectively, 5-98 and 8901 transfer-gen plants, and wherein, swimming lane 4-8 is the transfer-gen plant of 104 kinds, and swimming lane 9-12 is the transfer-gen plant of 5-98 kind, and swimming lane 13-19 is the transfer-gen plant of 8901 kinds.
(ii) T
1Dna molecule hybridize for transfer-gen plant detects
To detect the T of the specific fragment that obtains the dreb gene about 724bp through above-mentioned PCR
0Plant for plant, obtain T
1For transfer-gen plant, extract the blade genomic DNA respectively, with digoxin (DIG, Roche) (dreb gene of this 724bp is that the genomic DNA with mouseearcress is a template to the dreb gene of mark 724bp, utilize primer P1 and P2PCR amplification to obtain) as probe, carry out dot blot and detect.This dot blot is analyzed, use the Bio-Rad Bio-Dot Microfiltration of company dot blot instrument to carry out a film, hybridize and detect by the DIG DNA Labeling and Detection Kit of Roche company, dreb gene is carried out the DIG mark with the PCR method.The dot blot result as shown in Figure 3, showing in most of transfer-gen plant has hybridization signal, it is strong signal that part point is wherein arranged, and has proved that foreign gene has been incorporated in the genome of wheat.But, the also visible hybridization signal of non-transgenic contrast, this explanation also exists similar DREB transcription factor or the higher dna sequence dna of autoploidy probably in the genome of wheat.Among the figure, "+" lists plasmid pBAC128F; " CK " lists the non-transgenic plant; Other list transfer-gen plant.
(iii) T
1PCR for transfer-gen plant detects
Because there is false positive in dot blot, therefore be necessary further checking.19 transgenic lines choosing strong signal from the result of dot blot carry out pcr amplification, the same T of amplification method
0For transfer-gen plant, show and have only 12 strains systems to have the special band of 724bp in this 19 strains system that the result verifies mutually with dot blot, has confirmed that finally these 12 strains are transgenic line, i.e. 104-20 in 104 kinds, 104-25,104-79,104-18; 8901-50 in 8901 kinds, 8901-51,8901-32; 5-98-69 in the 5-98 kind, 5-98-70,5-98-73,5-98-76; 99-92-63 in the 99-92 kind.Although may in the genome of wheat, there be similar dreb gene, because the difference of sequence fails to amplify in contrast the dna fragmentation of identical size.Wherein the PCR result of 18 transgenic lines as shown in Figure 4, among Fig. 4,1 shows plasmid pBAC128F; 2 show the non-transgenic plant; 3-20 shows transfer-gen plant: 104-20,104-25,8901-42,5-98-8,5-98-11,104-17 respectively, 5-98-69,5-98-73,8901-38,8901-50,8901-51,104-79,104-78,8901-45,8901-32,104-18,8901-43,99-92-63.
(iv) T
1Molecular hyridization for the RNA of transfer-gen plant is identified
T
1When growing to tillering stage, arid was handled after 5 days for transfer-gen plant 7 strains (104-20,104-25,5-98-69,104-18,8901-51,99-92-63 and 8901-32) and non-transgenic plant, and the total RNA of blade is extracted in sampling.Guanidine isothiocyanate method (Sambrook J is adopted in the extraction of the total RNA of plant, Fritsch EF, Maniatis T.Molecular Cloning-A Laboratory Manual.2nd ed.New York:Cold Spring Harbor laboratory Press, 1989).The total RNA of blade that extracts carries out electrophoresis with 1.5% Ago-Gel in 1 * TAE, electrophoresis result as shown in Figure 5, among the figure, 1 shows the total RNA of non-transgenic plant, 2-8 shows the total RNA of transfer-gen plant.
The RNA sample is diluted to 0.4mg/ml with 6 * SSC, sample places 65 ℃ of water-bath sex change 15min, put quenching on ice, the point film, hybridization and detection are analyzed with the DNA dot blot, wherein, probe is with digoxin (DIG, Roche) dreb gene of the 724bp of mark, the dreb gene of this 724bp are that the genomic DNA with mouseearcress is a template, utilize primer P1 and P2 pcr amplification to obtain.Results of hybridization shows that foreign gene can normal transcription in selected 7 transfer-gen plants.
4, the mensuration of wheat leaf blade free proline content
Proline has height moist as a kind of osmotic adjustment material, is a kind of anti-dehydrating agent, and is in close relations with the drought resistance of plant.In general, same kind, proline content is high more, and drought resistance is strong more.
Will be through the T of (i) PCR test positive of (1) of step 3
0Cultivate to seedling stage for the plant plantation, according to document (Xu Xiaofeng, Zhu are. the research of PA method in the wheat leaf, biotechnology .1997,7 (1): method 40～42) is measured the wheat leaf blade free proline content, concrete grammar is as follows: adopt the method for lab simulation drought condition, after wheat four leaves stop during the phase watering 5 days, take by weighing wheat leaf blade 0.1g, put into test tube after shredding, add 5ml 3% sulfosalicylic acid solution, lixiviate is 10 minutes in boiling water bath, is chilled to room temperature.Draw leaching liquor 2ml in another test tube, add 2ml water, 2ml glacial acetic acid and 4ml 2.5% acid ninhydrine solution (is that solvent is prepared with 3: 2 glacial acetic acids and 6mol/L phosphoric acid) developed the color 60 minutes to boiling water bath, were cooled to room temperature.Add 4ml toluene, vibration is with the extraction red material.After leaving standstill, draw toluene layer, get the OD value in 721 type spectrophotometer 520nm wavelength place colorimetrics.The making of calibration curve: compound concentration is the serial proline standard liquids of ten of 1～10 μ g/ml.Get 2ml leaching liquor and 2ml water in standard liquid 2ml and the 2ml 3% sulfosalicylic acid solution replacement sample determination, develop the color, extraction and colorimetric (wavelength 520nm), last drawing standard curve by said procedure.
Calculate the content of the free proline of wheat leaf blade according to the value that records: proline content (μ g/g)=C*2.5/W
In the formula: C-----is with the proline content (μ g) of the sample of calibration curve acquisition
W-----wheat leaf blade weight (g)
2.5---leaching liquor 3% sulfosalicylic acid volume (5ml) and mensuration time institute's sample thief liquid long-pending (2ml) ratio when extracting proline
T
1Show that for transfer-gen plant seedling leaf proline content measurement result the part transgenic line is significantly improved than non-transgenic contrast proline content, can improve more than 2 times.Fig. 6 only lists tangible 16 strains of proline content amplification.8901-66 wherein, 5-98-69,5-98-70, the proline content of strains such as 104-79 system compares according to having improved more than 2 times more than 1100 μ g/g.CK represents the contrast of corresponding not transformed variety among the figure.
5, the drought resisting effect of transfer-gen plant is identified
Can wheat breed drought resisting, and the drought resisting degree is much, though can identify in the relevant physiological index, whether it really has drought resistance, still needs the check by reality.Adopt the method for lab simulation drought condition, the 104-20 in 104 kinds, 104-25,104-79,104-18; 8901-50 in 8901 kinds, 8901-51,8901-32; 5-98-69 in the 5-98 kind, 5-98-73, the 99-92-63T in the 99-92 kind
1Stopped during the phase watering 15 days for wheat four leaves, recover then to water 10 days.The result shows these transgenic lines after stopping to water 15 days, and blade still shows green, and the contrast blade is chlorosis, dried-up then; After the rehydration 10 days, transgenic line rejuvenates, and contrast is then dead.Fig. 7 A and Fig. 7 B shown respectively stop to water 15 days after the growing state of non-transgenic plant and part transfer-gen plant, Fig. 7 C and Fig. 7 D have shown the non-transgenic plant of rehydration after 10 days and the growing state of part transfer-gen plant respectively.
Sequence table
<160>2
<210>1
<211>727
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
ggatcctgat?caatgaactc?attttcagct?ttttctgaaa?tgtttggctc?cgattacgag 60
cctcaaggcg?gagattattg?tccgacgttg?gccacgagtt?gtccgaagaa?accggcgggc 120
cgtaagaagt?ttcgtgagac?tcgtcaccca?atttacagag?gagttcgtca?aagaaactcc 180
ggtaagtggg?tttctgaagt?gagagagcca?aacaagaaaa?ccaggatttg?gctcgggact 240
ttccaaaccg?ctgagatggc?agctcgtgct?cacgacgtcg?ctgcattagc?cctccgtggc 300
cgatcagcat?gtctcaactt?cgctgactcg?gcttggcggc?tacgaatccc?ggagtcaaca 360
tgcgccaagg?atatccaaaa?agcggctgct?gaagcggcgt?tggcttttca?agatgagacg 420
tgtgatacga?cgaccacgga?tcatggcctg?gacatggagg?agacgatggt?ggaagctatt 480
tatacaccgg?aacagagcga?aggtgcgttt?tatatggatg?aggagacaat?gtttgggatg 540
ccgactttgt?tggataatat?ggctgaaggc?atgcttttac?cgccgccgtc?tgttcaatgg 600
aatcataatt?atgacggcga?aggagatggt?gacgtgtcgc?tttggagtta?ctaatattcg 660
atagtcgttt?ccatttttgt?actatagttt?gaaaatattc?tagttccttt?ttttagaatg 720
ggagctc 727
<210>2
<211>1706
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
aagcttcatt?ttctgctaca?gaagtgttgt?tctctgtaaa?gtaatcagaa?ggaatgtaat 60
caacagaata?aatctgctct?tgaatgtcct?ttgagttctg?caggataata?tctgacccct 120
catatttaac?ctgcaataag?aagactagct?aaaatcagat?gatgccaaga?acaggaacca 180
gctctaccac?tctacagcaa?aagcaaaaac?tactgttgtg?tgtgccaact?aaaatcaact 240
cagggattta?ccgggtcatt?tttgatctct?agaacttcag?agaaacattg?gagatgatcc 300
tcactcgtgc?ataagagctt?gttctcctgt?atatcaaaag?atgctttatc?tcgcaatgaa 360
aaagaaaaag?ctgaactagt?tcaattagta?caatgcttac?acagcttgag?caataccatc 420
tcaaaatcca?actctggagt?ctccaactca?ataattttag?gtgtagcttt?atccgcaatg 480
gatctccgtc?atccttcttc?gaatagatca?taacttcggt?tttcaactcg?gtattcgctt 540
aagcaccaaa?atttccaacc?caagtatgaa?tataagatct?aagcaacaat?cagaaatgga 600
aactgagaaa?acacaccaca?aatttcgaaa?aatctacaac?caatctcact?ataagaaaca 660
aaggaccgtt?gacagaaaca?gtcagcgaga?ctcaggaaat?tcgaaatttc?acctccagga 720
actgataata?tctagatcga?aggaacttta?cctcgtctga?gtaataaact?ccgagcgaag 780
agtcgtcgat?ttcaaaaact?cgatagtcca?cactgacgcg?gtcgggaacc?acgtcggaaa 840
ggaacttcga?caaagcagct?tcaataggca?aatttccgat?agggatacta?acattttcga 900
tcgagccaaa?tcggagacgg?tcttcttctc?cgttgtagac?gatgggtgcc?gggaaattat 960
caggagccgg?aagattgagg?aagcctaggg?tttcaaatac?gtgagaaggt?ggagtagaga 1020
agtaatcgat?gttgagacat?cgagttcgca?tcgtaatttt?ctagatccgt?cttgggagct 1080
cagactgtat?cagtgatgat?gatgatgatg?aagaagagaa?cgaattttga?aattggcggt 1140
tttgaatttt?taagaaatta?aaaaatatcc?cccgtcgatt?tcaagaggga?gatggagata 1200
ccaaagcaac?tctcgccact?tgtcgtcttt?taattttaat?tgagtacgtt?atgccgtttt 1260
aaatgttcaa?aacagcacac?agttgatagc?tgaattgatt?ttttcttttg?ccgttttgtt 1320
atatttaaac?aacacacagt?gcatttgcca?aataactaca?tgatgggcca?ataaacgtgg 1380
accgactaaa?actaaataat?agaagataca?tcgataggct?tctctaaaga?tcggataaaa 1440
gataatgtcg?catagccacg?tagagagcaa?ctggctgaga?cgtggcagga?cgaaacggac 1500
gcatcgtacg?tgtcagaatc?ctacagaagt?aaagagacag?aagccagaga?gaggtggttc 1560
ggccatatgt?catcgttctc?tctataaact?ttatggaact?ttgttctgat?tttctcagag 1620
acacgaaaag?aaagaaaaca?acactagaac?aaagagggtt?tgattgattc?acttgaaaaa 1680
gagaaaacac?agctttggaa?ggatcc 1706
Claims (10)
1, a kind of method that strengthens drought resistance of wheat, it is encoding gene insertion plant expression vector with the DREB transcription factor, obtain containing the recombinant expression carrier of the encoding gene of DREB transcription factor, this recombinant expression carrier is imported in wheat, obtain the wheat that drought resistance strengthens.
2, method according to claim 1 is characterized in that: the encoding gene of described DREB transcription factor derives from mouseearcress or paddy rice or corn or soybean or rape.
3, method according to claim 2 is characterized in that: the encoding gene of described DREB transcription factor derives from mouseearcress.
4, method according to claim 3 is characterized in that: the encoding gene of described DREB transcription factor has the dna fragmentation from 5 ' end the 612nd to 1320 bit base of Genbank Access No.AB013816.
5, method according to claim 4, it is characterized in that: in described recombinant expression carrier, starting the promotor that the encoding gene of described DREB transcription factor transcribes is the promotor of RD29B gene or promotor or SAG12 promotor or the corn Ubi promotor or the CaMM 35S promoter of RD29A gene.
6, method according to claim 5 is characterized in that: the promotor of the hydrophilic albumen RD29B of described mouseearcress gene has the dna fragmentation from 5 ' end the 85th to 1784 bit base of Genbank Access No.D013044.
7, method according to claim 6 is characterized in that: the recombinant expression carrier of the encoding gene of the described DREB of containing transcription factor is pBAC123 or pBAC128F.
8, according to arbitrary described method in the claim 1 to 7, it is characterized in that: in the described method, be to be explant with wheat children tassel or rataria, the recombinant expression carrier that will contain the encoding gene of DREB transcription factor imports wheat.
9, according to arbitrary described method in the claim 1 to 7, it is characterized in that: the recombinant expression carrier of the encoding gene of the described DREB of containing transcription factor imports wheat by particle bombardment.
10, according to arbitrary described method in the claim 1 to 7, it is characterized in that: described wheat is following kind: 8901, and 5-98,99-92 and Bao Feng 104.
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| CNB2005101048704A CN100347305C (en) | 2005-09-23 | 2005-09-23 | Method for improving drought resistance of wheat |
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| CNB2005101048704A CN100347305C (en) | 2005-09-23 | 2005-09-23 | Method for improving drought resistance of wheat |
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| CN100347305C CN100347305C (en) | 2007-11-07 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103397097A (en) * | 2013-08-07 | 2013-11-20 | 新疆农垦科学院 | Molecular marker related to drought resistance of wheat and application thereof |
| CN106065404A (en) * | 2016-06-07 | 2016-11-02 | 北京农业生物技术研究中心 | A kind of method improving drought resistance of maize |
-
2005
- 2005-09-23 CN CNB2005101048704A patent/CN100347305C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103397097A (en) * | 2013-08-07 | 2013-11-20 | 新疆农垦科学院 | Molecular marker related to drought resistance of wheat and application thereof |
| CN106065404A (en) * | 2016-06-07 | 2016-11-02 | 北京农业生物技术研究中心 | A kind of method improving drought resistance of maize |
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| Publication number | Publication date |
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| CN100347305C (en) | 2007-11-07 |
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