CN103232533A - Wheat drought-resistant gene TaASR1 and application thereof - Google Patents

Abstract

The invention relates to the field of plant genetic engineering and provides a wheat drought-resistant gene TaASR1, the nucleotide sequence of which is shown in SEQ ID NO:1. The invention constructs a TaASR1 gene plant expression vector, transforms tobacco with the method mediated by Agrobacterium tumefaciens, and obtains TaASR1 gene-transferred tobacco plants. The drought simulation experiments confirmed that the transgenic tobacco with TaASR1 gene had stronger drought tolerance than the control tobacco.

Description

A kind of wheat drought resistance gene TaASR1 and its application

technical field

The invention relates to the field of plant genetic engineering, in particular to a wheat drought-resistant gene TaASR1, and various applications of the TaASR1 gene in improving the drought-resistant ability of plants.

Background technique

Plants are sessile benthic organisms and must adapt to the environment to survive, so abiotic stress responses play a very important role in plants. Abiotic stresses include stresses caused by complex environmental conditions such as light, high or low temperature, freezing, drought, salinity, heavy metals, and hypoxia. Due to global climate change, these abiotic stresses are affecting the habitats of plants and increasing. In order to adapt to their growth environment, plants have developed defense mechanisms against abiotic stresses through long-term evolution. The occurrence and transmission of these defense mechanisms involve the participation of various genes. Genes encoding transcription factor proteins are an important class of genes involved in plant adversity stress.

In 1993, the ABA-stress-maturation response protein gene (ASR1) was first isolated from tomato as a drought stress-induced gene [Iusem ND, Bartholomew DM, Hitz WD and ScolnikPA (1993) Tomato (Lycopersicon esculentum) transcript induced by waterdeficit and ripening.Plant Physiol.102, 1353-1354.], and the research on this kind of gene has been carried out extensively since then. Studies have found that ASR genes widely exist in plants. So far, ASR genes have been isolated from potatoes, corn, pomelo, loblolly pine, lilies, rice, and grapes, but no ASR genes have been found in the model plant Arabidopsis [Carrari F, Fernie AR and Iusem ND(2004) Heard it through the grapevine? ABA and sugar cross-talk: the ASR story. Trends Plant Sci.9, 57-59.]. As detected by subcellular localization, ASR1 protein was expressed in the nucleus. Yeast one-hybrid experiments proved that this kind of protein can bind to DNA, and it was deduced that ASR protein might be a kind of transcription factor. Existing studies have shown that plant ASR genes can be induced by environmental stresses such as drought, high salinity, and cold, and participate in the ABA signaling pathway. The ASR gene Sodip22 isolated from sugarcane exists only in epidermal cells, and its expression level is significantly induced under the conditions of ABA and drought treatment; the ASR gene was also cloned in Ginkgo biloba, and it was treated with high salt, mannitol and ABA, and its expression level was significantly induced. The expression levels in roots, stems and leaves increased significantly. In addition, overexpression of ASR family genes can significantly improve plant tolerance to abiotic stresses such as cold, drought, and salt. It was confirmed in lily that the accumulation of ASR protein is closely related to the desiccation of pollen tubes, and the ASR gene LLA23 was cloned from lily, and expression analysis showed that the gene was induced by ABA, NaCl and drought, and overexpression of this gene in Arabidopsis could Significantly improve the tolerance of plants to high salt [Yang CY, Chen YC, Jauh GY and Wang CS. (2005) A Lily ASR protein involves abscisicacid signaling and confers drought and salt resistance in Arabidopsis. Plant Phys iol. 139, 836- 846.].

Wheat is one of the earliest cultivated and most important food crops in the world. It is a staple food in more than 40 countries in the world, accounting for 35% of the world's total population. Governments and scientists of various countries have always paid great attention to the genetic law and genetic improvement of wheat. Improving wheat quality, increasing yield and increasing resistance have always been the focus of crop improvement research. Transcriptional regulation is one of the important ways of regulating gene expression. Transcription factors have the characteristics of binding to cis-acting elements and regulating the expression of multiple downstream genes. Therefore, finding transcription factors related to wheat quality and resistance, and using genetic engineering to increase their expression levels to regulate the expression of related downstream genes is a more effective way to improve wheat quality and resistance. As a transcription factor, ASR gene widely exists in plants, and its function is mainly related to the response of plants to abiotic conditions (light, water, temperature) stress. However, no ASR gene has been reported in wheat. Therefore, cloning and identifying the ASR gene in wheat has important scientific research and application value for theoretical research and practical application of improving wheat stress resistance.

Contents of the invention

The object of the present invention is to provide a kind of isolated wheat ABA-stress-maturation response protein and the gene encoding the protein, which is also called wheat drought resistance gene (TaASR1) in the present invention, and the present invention also provides that the gene can be used in cultivating high drought resistance plants in the application.

Realize the technical scheme of the present invention is:

The amino acid sequence of the isolated wheat ABA-stress-maturation response protein provided by the invention is:

(1) A protein consisting of the amino acid sequence shown in SEQ ID No.2; or

or

(3) A protein derived from (1) whose amino acid sequence shown in SEQ ID No.2 has been added, deleted or replaced by one or more amino acids and has equivalent activity.

The gene encoding the above-mentioned protein belongs to the scope of the present invention, and the present invention lists a nucleotide sequence of the gene (wheat drought-resistant gene TaASR1) encoding the above-mentioned isolated wheat ABA-stress-maturation response protein, and the nucleotide sequence is as SEQ ID No.1 is shown.

The gene encoding the wheat ABA-stress-maturation response protein provided by the invention, the expression vector containing the gene and the host introduced with the expression vector can be used for cultivating highly drought-resistant plants, such as cultivating highly drought-resistant tobacco.

The present invention combines the EST sequence in the database with the RACE technology, and obtains a cDNA sequence of an ABA-stress-maturation response protein gene with a full length of 414bp through various bioinformatics prediction methods. The NCBI accession number is: HQ287799, which The nucleotide sequence is shown in SEQ ID No.1. The gene encodes a protein of 137 amino acids in total, with a predicted molecular weight of 15.3 kDa, and its amino acid sequence is shown in SEQ ID No.2. Sequence comparison analysis showed that the amino acid sequence encoded by the gene had two typical conserved regions, ABA/WDS conserved domain, a His-rich region and two Ala-rich regions. Therefore, this gene belongs to the typical ABA-stress-maturation response protein family and is a new ASR gene in wheat.

It should be clear that those skilled in the art can substitute, delete and/or add one or more amino acids based on the amino acid sequence disclosed in the present invention without affecting the biological activity of the protein. The sequence of the mutant of the protein is obtained if the origin is above 95%. Therefore, the present invention also includes derivative proteins with high homology and biological activity obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence shown in SEQ ID No.2.

The gene of the present invention includes the nucleotide sequence encoding the above-mentioned protein.

In addition, it should be understood that, in view of codon degeneracy and codon bias of species, those skilled in the art can use codons suitable for expression of specific species as needed.

The gene and protein of the present invention can be cloned or isolated from the wheat variety Zhongchunzhong, or obtained by sequence chemical synthesis.

The gene of the present invention can be linked to the downstream of the promoter of the expression vector to construct a recombinant expression vector capable of expressing the protein, and then the expression vector can be introduced into various hosts by genetic transformation to obtain a transformant transfected with the TaASR1 gene.

The invention introduces wheat drought-resistant gene TaASR1 into tobacco, and the drought-resistant phenotype and drought-resistant physiological indexes of transgenic plants are significantly improved, indicating that the TaASR1 gene can be used to improve crop drought resistance.

Technical route of the present invention is as follows:

Query and obtain wheat EST→wheat material processing→target gene amplification→gene expression analysis→experimental load construction→transgenic plant stress resistance phenotype and physiological analysis→research conclusion

Description of drawings

Figure 1 Multiple sequence alignment and conserved region description of amino acids encoded by the wheat drought-resistant gene TaASR1 (Note: boxes represent ABA/WDS motifs; asterisks represent myristoylation sites; double dashes represent possible nuclear localization signals).

Figure 2 The expression analysis of TaASR1 gene in different tissues (A) and PEG6000 (B), ABA (C), hydrogen peroxide (D) treatment (Note: R, root; S, stem; L, leaf; ST, stamen; P, pistil; LE, lemma).

Fig. 3 Schematic diagram of the construction of tobacco overexpression vector pCAMBIA1304-TaASR1.

Fig. 4 Phenotype analysis of TaASR1-transferred tobacco (Note: WT is wild type, VC is empty vector plant, OE is TaASR1 overexpression line; A is six-week-old seedlings, B is three-week-old seedlings, and C is survival rate calculation) .

Fig. 5 Germination rate and root length analysis of TaASR1 gene-transferred tobacco seeds (Note: WT is wild type, VC is the empty vector plant, OE is the TaASR1 overexpression line; A is the photo of each line grown on MS for 8 days; a is the germination rate statistics of each strain grown on MS for 8 days; B is the photo of each strain grown on MS+150mM mannitol medium for 8 days; b is each strain grown on MS+150mM mannitol medium Germination rate statistics for 8 days of growth; C is a photo of each strain growing on MS+300mM mannitol medium for 8 days; c is the germination rate statistics of each strain growing on MS+300mM mannitol medium for 8 days; D is the picture of root length of one-week-old seedlings grown on MS for one week; E is the picture of root length of one-week-old seedlings grown on MS+150mM mannitol medium for one week; F is the picture of one-week-old seedlings grown on MS+300mM The root length picture after one week of growth on the mannitol medium; G is the root length statistics of each strain after one week of growth on MS, MS+150mM mannitol, and MS+300mM mannitol).

Detailed ways

Example 1 Cloning and Bioinformatics Analysis of Wheat Drought Resistance Gene TaASR1

The expressed sequence tag sequence (EST) of wheat can be searched through the DFCI wheat database (http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=wheat). From this database, we found that three EST sequences (BF414974, CA695407, DR734766) belonged to the ABA-stress-maturation response protein (ASR) family and encoded the same ASR protein with a conserved ABA/WDS domain. Sequence analysis showed that the 5'-ends of the proteins encoded by these three ESTs were intact, but the 3'-ends were missing. Using the RACE technique, we obtained the 3'-end of the EST sequence. Through sequence splicing and PCR amplification, we obtained the complete sequence of the gene and named it TaASR1, as shown in SEQ ID No.1. Using databases or software such as Blast, ORFFINDER, and DNAMAN to analyze showed that the TaASR1 DNA we obtained contained a complete open reading frame (ORF). Through multiple sequence alignment analysis, it was determined that the protein encoded by the gene had the conserved domain and active region of the ASR family proteins (Fig. 1). At the N-terminus of the sequence, there is a typical six-histidine disability capable of binding zinc; at the C-terminus, there are two typical leucine-rich regions, an ABA/WDS domain and a putative nuclear localization signal (see picture 1). These results indicated that TaASR1 obtained by us is a new wheat ASR member, which laid the foundation for the functional study of this gene.

Experimental steps:

1. Primer design: Design a RACE primer with Primer Premier5 based on the obtained EST fragment

P1UP: 5'-GAAGCACGAGGCGAAGAAGGACCC-3', the linker primer for RACE reaction is P1DOWN: 5'-CTAATACGACTCACTATAGGGC-3'.

2. Reaction system: Add 1.0 μL of cDNA template, 2.5 μL of 10×Buffer, 0.5 μL of dNTP, 1.0 μL of P1UP (10pM), 1.0 μL of P1DOWN (10pM), Taq polymerase (5u/μL) into a 0.2mL centrifuge tube 0.3 μL, ddH2O 18.7 μL.

3. Reaction program: 94°C, 5m; 94°C, 30s; 48°C, 30s; 72°C, 30s; 72°C, 10m; 35Cycle.

4. Recovery of target fragments: recovery with TIANGEN's agarose gel DNA recovery kit (refer to the instructions).

5. Connect the recovered product with pMD18-T vector (refer to the TaKaRa manual): add 0.5 μL of pMD18-T vector (50ng/μL) to a 0.2mL centrifuge tube in sequence, recover 3.0 μL of the product (100ng/μL), Solution I5. 5 μL, ddH2O 1.0 μL. After mixing, centrifuge briefly, collect the droplets on the tube wall to the bottom of the tube, and connect at 16°C for 12 hours.

6. Preparation of E.coli DH5α competent cells: refer to the Molecular Cloning Experiment Guide (Huang Peitang 2002).

7. Transformation of the ligation product into E.coli DH5α: refer to the Molecular Cloning Experiment Guide (Huang Peitang 2002).

8. Mini-extraction of recombinant plasmid DNA: refer to the Molecular Cloning Experiment Guide (Huang Peitang 2002).

9. PCR identification of the recombinant plasmid: The PCR reaction system is the same as in Example 1.2, and the reaction procedure is the same as in Example 1.3.

10. Recombinant plasmid double enzyme digestion identification and sequencing: add 5.0 μL of recombinant plasmid (900ng/μL), EcoR I (15u/μL) 1.0 μL, HindIII (15u/μL) 1.0 μL, 10×bufferK2 into a 0.2 mL centrifuge tube .0 μL, ddH2O 13.0 μL, mix well, centrifuge slightly, incubate at 37°C for 3 hours, then electrophoresis the digested product in 1% agarose gel, combine with PCR identification results to determine whether there is a corresponding fragment insertion. Sequencing was performed by Shanghai Bioengineering Co., Ltd.

11. Full-length clone of TaASR1 cDNA

(1) Primer design: P2UP: 5-TCAGCCGGCGGCCATGGCGGAGG-3; P2DOWN: 5-GTGATCTAGCCGA AGTGGTGGT-3.

(2) Reaction system: Add 1.0 μL of cDNA template, 2.5 μL of 10×Buffer, 0.5 μL of dNTP, 1.0 μL of P2UP (10pM), 1.0 μL of P2DOWN (10pM), and Taq polymerase (5u/μL) into a 0.2mL centrifuge tube. ) 0.3 μL, ddH2O 18.7 μL.

(3) Reaction program: 94°C, 5m; 94°C, 45s; 52°C, 45s; 72°C, 45s; 72°C, 10m; 35 cycles.

(4) Recovery, ligation, transformation, plasmid extraction, identification, and sequencing of target fragments: the operations are the same as those in 4-10 of Example 1.

The expression analysis of embodiment 2 gene

Experimental steps:

1. Treatment of experimental materials: Triticum aestivum L.cv.Chinese Spring was the tested wheat variety treated with adversity and various signaling molecules. After the above seeds have been sterilized and sterilized, germinated seeds are obtained in a clean petri dish with sterile water as the culture medium, planted on MS medium, and cultivated under the conditions of 16/8 photoperiod and 25°C to obtain seeds. Seedling. The special stress simulation treatment was carried out on the 10-day-old seedlings.

(1) PEG simulated drought treatment: the seedlings were transplanted into the culture solution containing 20% PEG6000 solution and cultured for 24 hours, and samples were taken at the time points of treatment 2, 6, 12, and 24 hours respectively, and frozen in liquid nitrogen.

(2) Signal molecule treatment: Spray 100 μM abscisic acid and 10 mM hydrogen peroxide on the leaves of seedlings for 24 hours, take samples at 2, 6, 12, and 24 hours after treatment, and freeze them in liquid nitrogen.

(3) Tissue expression treatment: For the above-mentioned treatment materials, pistils, stamens and lemma parts of seedling roots, stems, leaves and flowers were cut and frozen in liquid nitrogen.

2. RNA extraction of processed samples: use the plant tissue RNA extraction kit provided by Tiangen Biotechnology Company to extract RNA. Carry out standard operation according to its instruction manual. The integrity of the extracted RNA was checked by agarose gel electrophoresis.

3. Reverse transcription of RNA: Use the Ferments reverse transcription kit to reverse transcribe RNA into cDNA. The reverse transcription steps are as follows:

(1) Add 0.1ng-5μg of total RNA, 1μl of oligo(dT)18 primer and add to 12μl with double distilled water, mix well and centrifuge, incubate in a water bath at 65°C for 5min, and then immediately put it on ice to cool.

(2) Add 4 μl of reaction buffer, 1 μl of RNase inhibitor, 2 μl of 10mM dNTP Mix, and M-MuLV reverse transcriptase (200u/μl) μl, mix and centrifuge, and incubate in a water bath at 42°C for 60 minutes.

(3) Inactivation at 70°C for 5 minutes.

4. Primer design for real-time fluorescent quantitative PCR (qRT-PCR): Primers are designed in the untranslated region, excluding the conserved region of the protein encoded by the gene. The products amplified by the primers were sequenced and analyzed to confirm the reliability of the primers.

P3(TaASR1)UP: 5′-GCTGTCCTACTGGTCGCATA-3′

P3(TaASR1)DOWN: 5′-AACGGTGATCTAGCCGAAGT-3′

P4(TaAct i n)UP: 5′-TGCTATCCTTCGTTTGGACCTT-3′

P4(TaAct i n)DOWN: 5′-AGCGGTTGTTGTGAGGGAGT-3′

5. Real-time fluorescent quantitative PCR (qRT-PCR) condition exploration: A cDNA template obtained was diluted 10 times according to the concentration gradient and 6 gradients were used for qRT-PCR analysis, and the functional relationship between the Ct value and the concentration gradient was obtained. The amplification efficiency of the primers was calculated according to the slope.

6. Relative expression analysis by qRT-PCR: the target gene (TaASR1) and internal reference gene (TaActin) were simultaneously amplified for each sample, and four technical replicates were set. According to the reaction program of pre-denaturation at 94°C for 3 min, denaturation at 94°C for 10 s, annealing at 55°C for 10 s, and extension at 72°C for 30 s, a total of 40 cycles of reaction procedures were used to amplify (the reaction programs of the four genes are consistent), and at each cycle Fluorescent signals are collected during the extension phase. After the reaction, do the melting curve analysis at 94°C-55°C. The Ct value obtained by amplification was calculated using the 2-ΔΔCt method, and compared with the untreated control sample. ΔΔCt = (CT, Target-CT, Actin) Time x - (CT, Target-CT, Actin) Time0.

In this example, wheat seedlings cultured for 10 days were treated with PEG6000, ABA and hydrogen peroxide, and then the leaf tissue was collected to extract RNA and reverse transcribe it into cDNA as a template. In addition, samples from different tissues were collected to extract RNA, which was reverse-transcribed into cDNA as a template. Using SYBR I as a fluorescent dye, a QPCR reaction system was established for expression analysis (see Figure 2). The results showed that the gene was expressed in roots, stems, leaves, stamens, pistils and lemmas, with the highest expression in roots. Moreover, this gene can be significantly induced by osmotic stress (PEG6000), ABA and hydrogen peroxide. These results indicated that the gene may be involved in stress response, which provided a feasible basis for the subsequent identification of the gene's stress resistance function.

The construction of embodiment 3 plant expression vectors

Build steps:

1. PCR amplification of the target gene: According to the cDNA sequence of TaASR1, use Primer Premier5 to design a pair of primers for PCR amplification.

P5UP: CATGCCATGGCGGAGGAGAAGCACCACC

P5DOWN: GGACTAGTGCCGAAGTGGTGGTGCTTCT

The amplification procedure and reaction system are the same as Step 11 of Example 1.

2. Recovery of target fragments: same as step 4 of Example 1.

3. Digestion of the target fragment: Add 20 μL of the recovered target fragment, 4 μL of Nco I (10u/μL), 4 μL of Spe I (10u/μL), 10×buffer K+0.1% BSA5+5 μL in a 0.2 mL centrifuge tube, Water 12 μL.

4. Recovery of target fragments again: same as step 4 of Example 1.

5. Acquisition of pCAMBIA1304 vector: extract the pCAMBIA1304 plasmid stored in Escherichia coli, and the extraction method is the same as step 8 of Example 1. The plasmid was digested with reference to step 3 of Example 3, and the digested plasmid was recovered with an agarose gel recovery kit, and the method was the same as step 4 of Example 1.

6. Ligation of target fragment and pCAMBIA1304 vector: 3 μL of pCAMBIA1304 vector, 3 μL of target fragment, 1 μL of T4 DNA ligase (350u/μL), 1 μL of T4buffer, 2 μL of ddH2O. After mixing, centrifuge briefly, collect the droplets on the tube wall to the bottom of the tube, and connect at 16°C for 12 hours.

7. Competent preparation, transformation, screening and identification of recombinant plasmids, and sequencing, the operation process is the same as steps 6 to 10 of Example 1.

In this embodiment, based on the pCAMBIA1304 vector, the cDNA fragment of TaASR1 that does not contain a stop codon is inserted into the downstream of the 35S promoter and the upstream of the GFP gene, so that TaASR1 and GFP form a fusion protein, and the expression is initiated by the 35S promoter (see Fig. 3). Electrophoretic detection, enzyme digestion, ligation and other reactions and sequencing analysis showed that we have successfully constructed the pCAMBIA1304-TaASR1 plant expression vector, which laid the foundation for subsequent transgenic research.

Obtaining of Example 4 Transforming TaASR1 Gene Tobacco Strains

Specific steps:

1. Preparation of LBA4404 Agrobacterium Competent Cells:

(1) A single colony of Agrobacterium LBA4404 was inoculated in 5 ml of YEB liquid medium containing 50 mg/L Str, and cultured overnight at 200 rpm in dark at 28°C;

(2) Take 2ml of bacterial liquid and inoculate it into 50ml of YEB liquid medium containing 50mg/L Str and continue shaking culture until the OD600 value is about 0.4;

(3) Place the cultured bacterial solution on ice for 30 minutes, and centrifuge at 5000 rpm for 5 minutes at 4°C until the upper medium is removed;

(4) Add 10ml of pre-cooled 20mM CaCl2 to the collected thalline precipitate to fully resuspend the thallus;

(5) Centrifuge at 5000rpm for 5min at 4°C, pour off the supernatant, and remove the liquid as much as possible;

(6) Dispense 1ml of pre-cooled 20mM CaCl2 resuspended bacteria into one tube per 100μl, quickly freeze in liquid nitrogen and store at -70°C.

2. Transformation of Agrobacterium with pCAMBIA1304-TaASR1/pCAMBIA1304 expression vector:

(1) Take a tube of prepared Agrobacterium competent cells and put them on ice to thaw;

(2) Add 1 μl of the plasmid to be transformed, mix gently, and ice-bath for 30 minutes;

(3) Freeze in liquid nitrogen for 1 minute, and then quickly place it in a 37°C water bath for 5 minutes;

(4) Add 500 μl of antibiotic-free YEB liquid medium into a centrifuge tube, and incubate in dark at 28° C. and 200 rpm for 3-5 hours;

(5) Centrifuge at 8000rpm for 1min, take out part of the upper medium, leave 50-100μl supernatant to resuspend the bacteria;

(6) Spread the bacterial solution on the YEB solid medium containing 50mg/L Kan and 50mg/L Str, and incubate in the dark at 28°C for 2-3 days, and a single colony will grow.

3. Identification of positive clones: identify positive clones by extracting plasmids from transformed colonies and amplifying target genes by PCR. The primers are the same as step 1 of Example 3, and the amplification procedure and reaction system are the same as step 11 of Example 1.

4. Genetic transformation and regeneration of tobacco: Transformation of tobacco by Agrobacterium-mediated leaf disc transformation (Horsch et al.1985).

5. PCR detection of positive plants: extract the DNA of the transformed plants, use the target gene and GFP gene sequence to design primers, and carry out PCR amplification to screen positive plants.

P6(TaASR1) UP: GCACCACCACCACCTGTTCCAC

P6(TaASR1)DOWN: GCTCCGGGTCCTTCTTCGCCTC

P7(GFP)UP: TGGAGAGGGTGAAGGTGA

P7(GFP)DOWN: CTGGTAAAAGGACAGGGC

In this example, the genetic transformation method mediated by Agrobacterium tumefaciens was used to transform the pCAMBIA1304-TaASR1 expression vector into tobacco, and the T0 generation resistant tobacco plants were obtained through hygromycin culture and screening, and the target gene and GFP gene were detected by PCR to confirm the positive plants. The seeds of the resistant tobacco plants of the T0 generation were harvested and cultivated to obtain the T1 generation. The T1 positive plants were screened, the seeds were harvested, and cultivated to obtain the T2 generation. Continue to test the T2 generation plants to confirm that the target gene has not been segregated and lost, and finally obtain positive homozygous transgenic tobacco containing TaASR1 stable inheritance.

Example 5 Analysis of Drought Resistance Phenotype and Measurement of Related Physiological Indexes of Tobacco Lines Transduced with TaASR1 Gene

1. Analysis of drought resistance phenotype of TaASR1 gene-transferred tobacco lines: germinate wild-type, empty vector (pCAMBIA1304) and transgenic tobacco seeds (pCAMBIA1304-TaASR1) on MS medium, and transplant to sandy soil for culture after one week of germination in the matrix. Under normal light and water supply conditions, cultivate for two or five weeks. Three-week and six-week-old tobacco seedlings were deprived of water supply for 20 or 30 days, respectively, and the phenotypes of transgenic plants and wild-type were observed. Then, water it again for about a week, and then observe its phenotype. The results showed that after 20 days of drought treatment, the degree of curling of the leaves of the transgenic plants for six weeks was significantly lower than that of the control plants (see FIG. 4A ). Moreover, after 30 days of drought treatment, the three-week survival rate of transgenic plants was significantly higher than that of control plants (see Figures 4B and 4C). In addition, after 7 days of rehydration treatment after drought stress, the survival rate of the transgenic plants was still higher than that of the control plants (see FIG. 4B ). These results indicated that the transgenic plants were more resistant to drought stress than the control plants.

2. Analysis of resistance to osmotic stress of tobacco lines transfected with TaASR1 gene:

A. Germination rate analysis: surface disinfection of wild-type, empty carrier and transgenic tobacco seeds, and then sow them on MS and MS medium containing 150 or 300mM mannitol to count the germination of different lines at different times Rate. The results showed that the germination rate of the transgenic plants on the infiltration medium with different concentrations of mannitol was significantly higher than that of the control (see Figure 5).

B. Root length analysis: surface sterilize the wild-type, empty vector and transgenic tobacco seeds, then sow them on MS medium, and transfer them to MS or MS medium containing 150mM and 300mM after one week . After one week, the root length of each line was accurately measured. The results showed that the root length of the transgenic plants on the infiltration medium with different concentrations of mannitol was significantly longer than that of the control (see Figure 5). These results proved that under different degrees of osmotic stress, the transgenic plants showed better growth status than the control plants in the process of seed germination and root elongation. Based on the above phenotype analysis, the transgenic plants are obviously more resistant to drought and osmotic stress than the control plants, indicating that the gene has a certain application value in the improvement of crop resistance.

Claims (13)

1. the wheat ABA-of a separation coerces-ripe response protein, and its aminoacid sequence is:

(1) protein of being formed by the aminoacid sequence shown in the SEQ ID No.2; Or

(2) amino acid sequence homology that limits with sequence SEQ ID No.2 is at the aminoacid sequence of 80-100% coding identical function protein; Or

(3) aminoacid sequence shown in the SEQ ID No.2 is through increasing, lack or replace one or more amino acid and having the albumen of being derived by (1) with isoreactivity.

2. the Drought-resistance in Wheat gene (TaASR1) of coding claim 1 described albumen.

3. the described gene of claim 2, its nucleotide sequence is shown in SEQ ID No.1.

4. a recombinant expression vector is made of the plant expression vector that contains claim 2 or 3 described genes.

5. recombinant expression vector according to claim 4 is characterized in that, described plant expression vector is the pCAMBIA1304 carrier.

6. the Agrobacterium LBA4404 that contains claim 4 or 5 described recombinant expression vectors.

7. following one of every application in cultivating high drought-resistant plant:

(1) claim 2 or 3 described genes;

(2) claim 4 or 5 described recombinant expression vectors;

(3) contain the Agrobacterium LBA4404 of claim 4 or 5 described recombinant expression vectors.

8. application according to claim 7 is characterized in that, described plant is tobacco.

9. a cultivation has the method for the transgenic plant of high drought resistance, is with in claim 2 or the 3 described gene transferred plants.

10. method according to claim 9 is characterized in that, described gene is to import in the plant by recombinant expression vector, and described recombinant expression vector is made of the plant expression vector that contains claim 2 or 3 described genes.

11. method according to claim 10 is characterized in that, described plant expression vector is the pCAMBIA1304 carrier.

12., it is characterized in that described plant is tobacco according to claim 9,10 or 11 described methods.

13. method according to claim 12 is characterized in that, is by agriculture bacillus mediated leaf disc transformation method transformation of tobacco.

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