Application of wheat TaHAI3 gene or encoding protein thereof in regulation and control of plant drought resistance

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a wheat TaHAI3 gene or an encoding protein thereof in regulation and control of plant drought resistance. The function of the wheat TaHAI3 gene is researched through molecular biology and genetics methods, and it is found that after the TaHAI3 gene in wheat is knocked out, the drought resistance of wheat is remarkably improved; the TaHAI3 gene is transformed into wheat in an overexpression mode, and the drought resistance of a transgenic plant with overexpressed TaHAI3 is obviously reduced compared with that of a wild type plant. It is proved that the wheat TaHAI3 gene negatively regulates the drought resistance of wheat, and a new gene resource is provided for wheat drought-resistant molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to wheat. TaHAI3 Application of genes or their encoded proteins in regulating plant drought resistance. Background Technology

[0002] Wheat is one of the world's most important food crops and a major source of energy for humankind. However, drought stress severely restricts wheat yield and quality, causing huge economic losses. Discovering drought-resistant genes, elucidating their molecular mechanisms of drought response, and improving wheat traits are of great practical significance for increasing wheat yield and ensuring food security. Therefore, it is urgent to discover drought-resistant genes in wheat and cultivate new drought-resistant wheat germplasm.

[0003] TaHAI3 The gene encodes a PP2C protein phosphatase, primarily responsible for removing phosphate groups from proteins (dephosphorylation), which can shut down many signaling pathways activated by kinases. When drought stress occurs, plants synthesize ABA, which binds to its receptors such as PYLs, enhancing wheat drought resistance by inhibiting stomatal opening. However, this technology has not yet been observed... TaHAI3 Reports on the application of genes in drought resistance. Summary of the Invention

[0004] The purpose of this invention is to provide wheat TaHAI3 The application of genes or their encoded proteins in regulating plant drought resistance provides technical support for the breeding and improvement of drought-resistant plant varieties.

[0005] This invention provides wheat TaHAI3 The application of genes or their encoded proteins in regulating plant drought resistance and / or cultivating drought-resistant plants; the wheat TaHAI3 The amino acid sequence encoded by the gene is shown in SEQ ID NO:2.

[0006] Preferably, the application of regulating plant drought resistance includes: positively regulating the wheat TaHAI3 The application of genes or their encoded proteins in reducing plant drought resistance, and / or negatively regulating the wheat TaHAI3 Application of genes or their encoded proteins in improving plant drought resistance.

[0007] This invention provides negative regulation of wheat TaHAI3 The application of first biological materials expressing genes or their encoded proteins in improving plant drought resistance, specifically wheat. TaHAI3 The amino acid sequence encoded by the gene is shown in SEQ ID NO:2.

[0008] Preferably, the first biomaterial includes any one or more of the following: A) Knockout wheat TaHAI3 Nucleic acid molecules of genes; B) A recombinant vector containing the nucleic acid molecules described in A); C) Engineered bacteria containing the nucleic acid molecules described in A); D) Engineered bacteria containing the recombinant vector described in B).

[0009] Preferably, the nucleic acid molecule includes target site 1 and target site 2; the nucleotide sequences of target site 1 and target site 2 are shown in SEQ ID NO:13 and SEQ ID NO:14, respectively.

[0010] This invention also provides positive regulation of wheat TaHAI3 The application of second biological materials expressing genes or their encoded proteins in reducing plant drought resistance, such as wheat. TaHAI3 The amino acid sequence encoded by the gene is shown in SEQ ID NO:2.

[0011] Preferably, the second biomaterial includes any one or more of the following: 1) Contains wheat TaHAI3 Expression cassettes of gene coding regions; 2) Contains wheat TaHAI3 Recombinant vectors for gene coding regions; 3) A recombinant vector containing the expression cassette described in 1); 4) Contains wheat TaHAI3 Engineered bacteria that control the gene coding region; 5) Engineered bacteria containing the expression cassette described in 1); 6) Engineered bacteria containing the recombinant vector described in 2) or 3); The wheat TaHAI3 The nucleotide sequence of the gene coding region is shown in SEQ ID NO:1.

[0012] Preferably, the plant includes wheat.

[0013] This invention also provides a method for breeding drought-resistant wheat varieties, comprising the following steps: reducing the expression level of TaHAI3 protein in recipient wheat or inhibiting the expression of TaHAI3 protein in recipient wheat. TaHAI3 Gene expression was used to obtain the drought-resistant wheat variety. The wheat TaHAI3 The gene encodes the TaHAI3 protein, the amino acid sequence of which is shown in SEQ ID NO:2.

[0014] This invention also provides a method for breeding wheat varieties with reduced drought resistance, comprising the following steps: increasing the expression level of TaHAI3 protein in recipient wheat or promoting the expression of wheat protein in recipient wheat. TaHAI3Gene expression was used to obtain wheat varieties with reduced drought resistance. The wheat TaHAI3 The gene encodes the TaHAI3 protein, the amino acid sequence of which is shown in SEQ ID NO:2.

[0015] Beneficial effects: This invention provides wheat TaHAI3 The application of genes or their encoded proteins in regulating plant drought resistance and / or cultivating drought-resistant plants; the wheat TaHAI3 The amino acid sequence encoded by the gene is shown in SEQ ID NO:2. This invention studies wheat using molecular biology and genetic methods. TaHAI3 The function of the gene was discovered, which is related to wheat. TaHAI3 After gene knockout, wheat drought resistance was significantly improved; the gene was overexpressed to... TaHAI3 When the gene was transformed into wheat, overexpression was found. TaHAI3 The transgenic plants of the gene showed significantly reduced drought resistance compared to the wild type. This demonstrates the effectiveness of the wheat... TaHAI3 The negative regulation of drought resistance by genes provides new genetic resources for molecular breeding of drought-resistant wheat. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Picture 1 Wheat in Example 2 TaHAI3 Image showing the results of gene overexpression level identification; Picture 2 In Example 3 TaHAI3 The diagram shows the gene editing status of knockout mutants, where CR-1 and CR-2 are two independent CRISPR gene editing lines; Picture 3 For example, the wild-type Fielder in Example 4, TaHAI3 Overexpressing plants and TaHAI3 Phenotypes of knockout mutants before and after drought stress; Picture 4 For example, the wild-type Fielder in Example 4, TaHAI3 Overexpression plants and TaHAI3 Statistical results of aboveground fresh weight of knockout mutants under drought stress; Picture 5 For example, the wild-type Fielder in Example 4, TaHAI3 Overexpression plants and TaHAI3 A statistical chart showing the survival rate of knockout mutants after drought and rehydration; Pictures 4~5 middle" "express P＜0.05 ; "express P＜0.01 . Detailed Implementation

[0018] This invention provides wheat TaHAI3 The application of genes or their encoded proteins in regulating plant drought resistance and / or cultivating drought-resistant plants; the wheat TaHAI3 The amino acid sequence encoded by the gene is shown in SEQ ID NO:2.

[0019] In the present invention, SEQ ID The amino acid sequence shown in NO:2 is as follows: MSMAQVCCDSASSAVVVGAEAEARARARAGRRRRAGDAAVARWKVTAEAPQGAEEAAATRKRRAAGGEGVAAKRHGFTSVAGRRREMEDAVSIREAFTAPAEEGKPRRDFYGVFDGHGCSHVADACRERMHELVAEELAGAAQPESWTGAMERSFARMDAEVTASGGG DSASCRCEAN

[0020] As one implementation method, the wheat TaHAI3 TaHAI3 The nucleotide sequences of the upstream and downstream primers of the primer pair for the gene coding region are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0021] As one implementation method, the application of regulating plant drought resistance includes: positively regulating the wheat TaHAI3 Application of genes or their encoded proteins in reducing plant drought resistance, and / or negative regulation of wheat TaHAI3 Application of genes or their encoded proteins in improving plant drought resistance.

[0022] This invention provides negative regulation of wheat TaHAI3 The application of first biological materials expressing genes or their encoded proteins in improving plant drought resistance, specifically wheat. TaHAI3 The amino acid sequence encoded by the gene is shown in SEQ ID NO:2. As one embodiment, the wheat... TaHAI3 The nucleotide sequence of the gene coding region is shown in SEQ ID NO:1.

[0023] In one embodiment, the first biomaterial includes any one or more of the following: A) Knockout wheat TaHAI3 Nucleic acid molecules of genes; B) A recombinant vector containing the nucleic acid molecules described in A); C) Engineered bacteria containing the nucleic acid molecules described in A); D) Engineered bacteria containing the recombinant vector described in B).

[0024] In one embodiment, the nucleic acid molecule includes target site 1 and target site 2; the nucleotide sequences of target site 1 and target site 2 are shown in SEQ ID NO:13 and SEQ ID NO:14, respectively. In one embodiment, the starting vector in the recombinant vector is a plasmid vector; in another embodiment, the plasmid vector is a pBUE411 plasmid vector. In one embodiment, the initial strain of the engineered bacteria is Agrobacterium; in another embodiment, the Agrobacterium is Agrobacterium EHA105. This invention does not specifically limit the construction method of the recombinant vector and engineered bacteria, and uses conventional methods for constructing recombinant expression vectors and engineered bacteria in the art.

[0025] This invention also provides positive regulation of wheat TaHAI3 The application of second biological materials expressing genes or their encoded proteins in reducing plant drought resistance, such as wheat. TaHAI3 The amino acid sequence encoded by the gene is shown in SEQ ID NO:2.

[0026] In one embodiment, the second biomaterial includes any one or more of the following: 1) Contains wheat TaHAI3 Expression cassettes of gene coding regions; 2) Contains wheat TaHAI3 Recombinant vectors for gene coding regions; 3) A recombinant vector containing the expression cassette described in 1); 4) Contains wheat TaHAI3 Engineered bacteria that control the gene coding region; 5) Engineered bacteria containing the expression cassette described in 1); 6) Engineered bacteria containing the recombinant vector described in 2) or 3); The wheat TaHAI3 The nucleotide sequence of the gene coding region is shown in SEQ ID NO:1.

[0027] In one embodiment, the starting vector in the recombinant vector can be a plasmid vector; in another embodiment, the plasmid vector is pENTR, pCAMBIA1300-EGFP, pCAMBIA1307-FLAG, pWMB110, pCUB, or pC336. In one embodiment of the present invention, pCUB is used as the starting vector to recombinant wheat... TaHAI3 Genes are connected to Bam At position HI, the recombinant vector used in the embodiments of the present invention is constructed. As one embodiment, the recombinant vector of the present invention is used in wheat... TaHAI3 The transcription initiation nucleotide of the gene may also contain a constitutive promoter or an enhancing promoter to promote the wheat TaHAI3 Gene overexpression. In one embodiment, the constitutive or enhanced promoter described in this invention can be the maize ubiquitin promoter or the cauliflower mosaic virus 35S promoter. In one embodiment, the starting strain in the engineered bacteria is Agrobacterium; in another embodiment, the Agrobacterium is Agrobacterium EHA105. This invention does not specifically limit the construction methods of the recombinant vector and engineered bacteria; conventional methods for constructing recombinant vectors and engineered bacteria in the art can be used.

[0028] In one embodiment, the plant described in this invention is a monocotyledonous plant; in another embodiment, the monocotyledonous plant is a grass; in yet another embodiment, the grass is wheat.

[0029] This invention also provides a method for breeding drought-resistant wheat varieties, comprising the following steps: reducing the expression level of TaHAI3 protein in recipient wheat or inhibiting the expression of TaHAI3 protein in recipient wheat. TaHAI3 Gene expression was used to obtain the drought-resistant wheat variety; the wheat TaHAI3The gene encodes the TaHAI3 protein, the amino acid sequence of which is shown in SEQ ID NO:2.

[0030] As one implementation method, the method involves increasing the expression level of TaHAI3 protein in recipient wheat or promoting the expression of wheat protein in recipient wheat. TaHAI3 Methods of gene expression include: knocking out the wheat TaHAI3 The gene's nucleic acid molecules are introduced into recipient wheat; as another embodiment, the knockout wheat is... TaHAI3 The recombinant expression vector of the gene's nucleic acid molecule was transferred into the recipient wheat.

[0031] This invention also provides a method for breeding wheat varieties with reduced drought resistance, comprising the following steps: increasing the expression level of TaHAI3 protein in recipient wheat or promoting the expression of wheat protein in recipient wheat. TaHAI3 Gene expression was used to obtain wheat varieties with reduced drought resistance. The wheat TaHAI3 The gene encodes the TaHAI3 protein, the amino acid sequence of which is shown in SEQ ID NO:2.

[0032] As one implementation method, the wheat TaHAI3 The nucleotide sequence of the gene coding region is shown in SEQ ID NO:1. As one implementation, this method involves increasing the expression level of TaHAI3 protein in recipient wheat or promoting the expression of wheat protein in recipient wheat. TaHAI3 Methods of gene expression include: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] TaHAI3 The nucleotide sequence of the gene coding region is introduced into recipient wheat; as another embodiment, the wheat containing the gene coding region is introduced into recipient wheat. TaHAI3 The recombinant expression vector of the nucleotide sequence of the gene coding region was transferred into the recipient wheat.

[0033] To further illustrate the present invention, the technical effects provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1 wheat TaHAI3 Cloning of genes Obtained from WheatOmics 1.0 database TaHAI3 Sequence retrieval TaHAI3 Design specific primers based on ORF sequences, as follows: TaHAI3- F (SEQ ID NO:3): 5'-ATGTCGATGGCGCAGGTGT-3'; TaHAI3-R (SEQ ID NO:4): 5'-CTACAAATGATTCCTTGG-3'.

[0035] Using leaves of the wheat variety Fielder as templates, RNA was extracted using the Trizol method (Thermo Fisher Scientific, catalog number: 15596026CN), and cDNA was obtained using a reverse transcription kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number: R222-01).

[0036] 1. RNA extraction, the steps are as follows: (1) Take an appropriate amount of seedling leaves of the wheat variety Fielder and put them into a pre-cooled 2.0 mL EP tube, add steel balls, and grind the leaves into powder at 1200 rpm for 60 s on a grinder; (2) Add 1 mL Trizol, mix by inversion, let stand at room temperature for 5 min, then centrifuge at 12000 rpm for 10 min at 4℃; (3) Transfer the supernatant to a new 1.5 mL EP tube, add 200 μL of chloroform, shake for 20 s, and let stand at room temperature for 3 min; (4) Centrifuge at 12,000 rpm for 10 min at 4℃; transfer 400 μL of supernatant to a new 1.5 mL EP tube; (5) Add 400 μL of isopropanol, mix by inversion, place at room temperature for 10 min, and centrifuge at 12000 rpm for 10 min at 4℃. (6) Discard the supernatant and add 1 mL of 75% ethanol solution to wash the precipitate; (7) Discard the supernatant and add 1 mL of 75% ethanol solution to wash the precipitate; (8) Centrifuge at 7500 rpm for 5 min at 4℃, and discard the supernatant; (9) After drying at room temperature, add 50 μL of DEPC water to dissolve and measure the concentration.

[0037] 2. RNA reverse transcription to prepare cDNA, the steps are as follows: (1) Take 1 μg of RNA obtained from step 1 and add RNase-free ddH2O to make up to 12 μL. Then add 4×g DNA wiper Mix and mix well. Then react at 42℃ for 2 min in a PCR instrument. (2) Add 4 μL of HiScriptII qRT SupermixII, mix well, and set the temperature gradient to 50℃ for 15 min, 85℃ for 5 min, and 4℃ for 10 min to obtain the reaction product, which is cDNA; use TaHAI3Using specific primers, PCR amplification was performed with the above cDNA as a template. After sequencing and alignment, the result was shown in SEQ ID NO:1. TaHAI3 Nucleotide sequence.

[0038] PCR reaction program: 94℃ pre-denaturation for 2 min; 98℃ for 10 s, 58℃ for 15 s, 68℃ for 1 min, 35 cycles; 68℃ extension for 5 min.

[0039] Example 2 TaHAI3 Obtaining transgenic plants through overexpression Using restriction endonucleases Bam The pCUB vector was digested with HI single enzyme at 37℃, and then expressed according to the pCUB overexpression vector. Bam Design specific adapter primers for HI restriction sites. TaHAI3 The gene is amplified to obtain a PCR product with an adapter.

[0040] The specific primers are as follows: TaHAI3 -pCUB-F: 5'-CAGGTCGACTCTAGAGGATCC ATGTCGATGGCGCAGGTG-3' (SEQ IDNO: 5); TaHAI3 -pCUB-R: 5'-AGCTCGTACCCGGGGATCC CAAATGATTCCTTGGCCG-3' (SEQ IDNO: 6); The PCR products were ligated to the pCUB restriction vector fragment using the seamless ligation kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number: C112-01 / 02) and transformed into Escherichia coli.

[0041] The transformation method for E. coli is as follows: Competent E. coli cells were placed on ice and allowed to thaw completely. 20 ng of the constructed vector was added, and the mixture was incubated on ice for 30 min. After the ice bath, the cells were heat-shocked in a 42°C water bath for 50 s, then immediately placed on ice. After 3 min, 400 μL of sterile LB broth was added, and the mixture was incubated at 37°C for 200 rpm for 45 min. 100 μL of the bacterial culture was aspirated in a clean bench and evenly spread onto LB agar plates containing kanamycin using a sterile spreader. The plates were then incubated at 37°C for 12–16 h. Single colonies were picked, and plasmids were extracted for PCR identification and sequencing.

[0042] The plasmids were transformed into Agrobacterium EHA105, and the wheat variety Fielder was transformed using Agrobacterium-mediated gene transformation. T0 generation transgenic plants were then identified by PCR.

[0043] The PCR identification primers are: pCUBF: 5'-GTATTAAATGTATAATTGCGGGACTCTAA-3' (SEQ ID NO: 7); pCUBR: 5'-TGTTTCTTTTGTCGATGCTCACC-3' (SEQ ID NO: 8); Positive transgenic plants were planted in a 22°C constant-temperature artificial climate chamber. After cultivation to harvest, T1 generation transgenic plants were planted using the same method, and positive plants were identified by PCR. This process was repeated until the T2 generation transgenic plants were completely homozygous. TaHAI3 The expression level was detected, and the expression level was... TaHAI3 The primers and internal reference primers used to detect the expression level are as follows: The primers used for quantitative analysis are: TaHAI3 -qrt-F: 5'-CAGGAGGGAGATGGAGGAC-3' (SEQ ID NO: 9); TaHAI3 -qrt-R: 5'-TCCAGGACTCGGGCTGT-3' (SEQ ID NO: 10); Actin-F: 5'-GACCGTATGAGCAAGGAGAT-3' (SEQ ID NO: 11); Actin-R: 5'-CAATCGCTGGACCTGACTC-3' (SEQ ID NO: 12).

[0044] Two homozygous transgenic lines with increased expression levels, OE-1 and OE-2, were selected for subsequent experiments.

[0045] TaHAI3 The method for detecting the expression level in transgenic plants is as follows: Transgenic plants were selected from the OE-1 and OE-2 transgenic lines. RNA was extracted from leaves of both transgenic plants and the wild-type material (WT) and reverse transcribed into cDNA. Expression levels were detected using real-time quantitative primers. The amplification program was as follows: 94℃ pre-denaturation for 3 min; 95℃ for 15 s, 60℃ for 15 s, 72℃ for 30 s, 40 cycles; melting curves were calculated and plotted at 65℃-94℃ for detection. The average of three replicates was used to represent the results. TaHAI3 The relative expression levels of genes, the results are as follows: Picture 1 As shown.

[0046] Example 3 TaHAI3 CRISPR knockout plants obtained according to TaHAI3 From the three conserved regions of the partial homologous genes, sequences containing the PAM domain of NGG were selected, and two target sites were chosen, including: Target site 1: 5'-GTGCTGTGACTCGGCCTCGTCGG-3' (SEQ ID NO:13); Target site 2: 5'-AAACGCCACGGGTTCACGTCGG-3' (SEQ ID NO: 14).

[0047] Design knockout primers based on target site 1 and target site 2: TAHAI3-P411-F: 5'-AATAATGGTCTCAAGCGTGCTGTGACTCGGCCTCGT-3' (SEQ ID NO: 15); TAHAI3-P411-F0: 5'-GTGCTGTGACTCGGCCTCGTGTTTTAGAGCTAGAAATAGC-3' (SEQ IDNO: 16); TAHAI3-P411-R0: 5'-ACGTGAACCCGTGGCGTTTCGCTTCTTGGTGCC-3' (SEQ ID NO: 17); TAHAI3-P411-R: 5'-ATTATTGGTCTCTAAACACGTGAACCCGTGGCGTTT-3' (SEQ ID NO: 18).

[0048] The pMT1T2 plasmid (disclosed in Chinese patent CN118931942A) was amplified by PCR using the above primers to obtain a DNA fragment containing the target site and vector adapter, and the target fragment was recovered by gel extraction.

[0049] use Bsa The pBUE411 vector was digested with restriction endonucleases, and the digested fragments were ligated with gel-recovered fragments using T4 ligase at 37°C for 5 hours. The ligation product was then transformed into competent E. coli cells, and positive plaques were identified by PCR. Plasmids were extracted and sequenced.

[0050] The correctly sequenced plasmid was transformed into Agrobacterium EHA105, and the wheat variety Fielder was transformed using Agrobacterium-mediated gene transformation. After obtaining positive plants, [further details needed]. TaHAI3Specific primers were designed at both ends of the three homologous gene target sites. Sanger sequencing technology was used to identify the gene-edited materials, and finally, two T2 generation independent knockout lines (CR-1 and CR-2) with different mutation types were selected. The mutation types are as follows: Picture 2 As shown.

[0051] The detection primers are shown below: TaHAI3-CRAF: 5'-TTAGATCGGACTTTCAACACAAC-3' (SEQ ID NO: 19); TaHAI3-CRAR: 5'-CGAGCTGTTCTTCGTCAGC-3' (SEQ ID NO: 20); TaHAI3-CRBF: 5'-GCGACTGCGCTTTATAAGC-3' (SEQ ID NO: 21); TaHAI3-CRBR: 5'-GTTGACCACGTCCCACAGA-3' (SEQ ID NO: 22); TaHAI3-CRDF: 5'-CGGCGATGGAAAGAATTTC-3' (SEQ ID NO: 23); TaHAI3-CRDR: 5'-CTCGTGCATCCGGTCTC-3' (SEQ ID NO: 24).

[0052] CR-1 strain TaHAI3A A 1 bp deletion at the left target site in the subgenome; TaHAI3B A 1bp deletion at the left target site in the subgenome; TaHAI3D A 196 bp deletion was found between subgenomic targets. The CR-2 strain... TaHAI3A The left target site in the subgenome was deleted by 8 bp; TaHAI3B A 1 bp deletion at the left target site in the subgenome; TaHAI3D The left target site in the subgenome is deleted by 1 bp.

[0053] Example 4 TaHAI3 Phenotypic analysis of drought resistance in transgenic plants Wild-type Fielder was treated with 1% sodium hypochlorite. TaHAI3 Overexpression (OE-1, OE-2) and TaHAI3CRISPR knockout mutant (CR-1, CR-2) seeds were washed for 10 min, rinsed with distilled water to remove sodium hypochlorite, and then neatly arranged in petri dishes lined with filter paper. 1 mL of distilled water was added, and the dishes were placed at room temperature. Once the seeds showed signs of germination, seedlings with uniform germination were selected and sown uniformly in potting soil (31 cm × 24 cm × 9 cm). Three biological replicates were set up and the plants were cultured in a light incubator (16 h light / 8 h dark, 16℃ / 14℃, 600 μmol / m² light intensity). -2 s -1 After the embryos reached the two-leaf-one-heart stage, watering was stopped (approximately 20 days). The overexpression and wild-type combinations were treated with drought for 18 days before rehydration, while the knockout mutant and wild-type combinations were treated with drought for 20 days before rehydration. Three days after rehydration, photographs were taken, survival rates were recorded, and the fresh weight of the aboveground parts was measured. A significant difference analysis was performed, and the results are as follows: Pictures 4~5 As shown.

[0054] The results showed that, compared with the wild type, TaHAI3 Overexpression plants exhibit a drought-sensitive phenotype. TaHAI3 Knockout plants exhibited a drought-tolerant phenotype. Picture 3 ), statistical results show that after drought and flooding TaHAI3 The survival rate of knockout mutants was significantly higher than that of wild-type, while the survival rate of overexpression plants was significantly lower than that of wild-type. Picture 4 ); TaHAI3 The aboveground fresh weight of knockout mutant plants was significantly higher than that of wild type, while the aboveground fresh weight of overexpression plants was significantly lower than that of wild type. Picture 5 ).

[0055] The above results indicate that TaHAI3 This gene negatively regulates wheat drought resistance; knocking out this gene can significantly improve wheat drought resistance, and this gene has broad application prospects in the genetic improvement of wheat drought resistance.

[0056] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Wheat TaHAI3 The application of genes or their encoded proteins in regulating plant drought resistance and / or cultivating drought-resistant plants; the wheat TaHAI3 The amino acid sequence encoded by the gene is shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The application of regulating plant drought resistance includes: positively regulating the drought resistance of wheat. TaHAI3 The application of genes or their encoded proteins in reducing plant drought resistance, and / or negatively regulating the wheat TaHAI3 Application of genes or their encoded proteins in improving plant drought resistance.

3. Negative regulation of wheat TaHAI3 The application of first biological materials expressing genes or their encoded proteins in improving plant drought resistance, specifically wheat. TaHAI3 The amino acid sequence encoded by the gene is shown in SEQ ID NO:

2.

4. The application according to claim 3, characterized in that, The first biological material includes any one or more of the following: A) Knockout wheat TaHAI3 Nucleic acid molecules of genes; B) A recombinant vector containing the nucleic acid molecules described in A); C) Engineered bacteria containing the nucleic acid molecules described in A); D) Engineered bacteria containing the recombinant vector described in B).

5. The application according to claim 4, characterized in that, The nucleic acid molecule includes target site 1 and target site 2; the nucleotide sequences of target site 1 and target site 2 are shown in SEQ ID NO:13 and SEQ ID NO:14, respectively.

6. Positive regulation of wheat TaHAI3 The application of second biological materials expressing genes or their encoded proteins in reducing plant drought resistance, such as wheat. TaHAI3 The amino acid sequence encoded by the gene is shown in SEQ ID NO:

2.

7. The application according to claim 6, characterized in that, The second biomaterial includes any one or more of the following: 1) Contains wheat TaHAI3 Expression cassettes of gene coding regions; 2) Contains wheat TaHAI3 Recombinant vectors for gene coding regions; 3) A recombinant vector containing the expression cassette described in 1); 4) Contains wheat TaHAI3 Engineered bacteria that control the gene coding region; 5) Engineered bacteria containing the expression cassette described in 1); 6) Engineered bacteria containing the recombinant vector described in 2) or 3); The wheat TaHAI3 The nucleotide sequence of the gene coding region is shown in SEQ ID NO:

1.

8. The application according to any one of claims 1 to 7, characterized in that, The plant mentioned includes wheat.

9. A method for breeding drought-resistant wheat varieties, characterized in that, The steps include: reducing the expression level of TaHAI3 protein in recipient wheat or inhibiting the expression of TaHAI3 protein in recipient wheat. TaHAI3 Gene expression was used to obtain the drought-resistant wheat variety. The wheat TaHAI3 The gene encodes the TaHAI3 protein, the amino acid sequence of which is shown in SEQ ID NO:

2.

10. A method for breeding wheat varieties with reduced drought resistance, characterized in that, The steps include: increasing the expression level of TaHAI3 protein in recipient wheat or promoting the expression of wheat protein in recipient wheat. TaHAI3 Gene expression was used to obtain wheat varieties with reduced drought resistance. The wheat TaHAI3 The gene encodes the TaHAI3 protein, the amino acid sequence of which is shown in SEQ ID NO:2.

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