Application of TaAP2-4 gene in regulating and controlling starch content of wheat grains

Abstract

The invention discloses an application of a TaAP2-4 gene in regulating and controlling starch content of wheat grains, which is used for enhancing the expression quantity of the TaAP2-4 gene to improve the starch content of the wheat grains and weakening the expression quantity of the TaAP2-4 gene to reduce the starch content of the wheat grains. The TaAP2-4 gene is located on a chromosome 4 of a wheat genome, and three copies, namely TaAP2-4-A, TaAP2-4-B and TaAP2-4-D, are formed on the chromosome 4. According to the invention, a Crispr / Cas9 gene editing vector for knocking out the TaAP2-4 gene is constructed by adopting a reverse genetics method, the TaAP2-4 gene is successfully knocked out in a wheat material by adopting an agrobacterium-mediated method, and the starch content of a knocked-out mutant is reduced, so that the condition that the TaAP2-4 gene plays a positive regulation role in a wheat grain starch synthesis process is known. In practical application, directional knockout mutation of the genome level TaAP2-4 gene can be achieved through a gene transformation process, the gene knockout mutation method can be used for verifying functions of the TaAP2-4 gene, and materials are provided for mining other starch synthesis related genes.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to a TaAP2-4 Application of genes in regulating starch content in wheat grains. Background Art

[0002] Wheat is one of the world's major grain crops, with an annual production exceeding 600 million tons. Wheat grain yield is primarily determined by photosynthesis and the partitioning of dry matter between tissues. Therefore, the ability of source tissues to produce photosensitized materials during grain filling, as well as the ability of the grain to convert and store these materials, are key factors limiting wheat yield. Starch is a key storage carbohydrate in wheat, comprising 65-70% of the grain's weight. Furthermore, the starch-glutenin interaction significantly impacts wheat grain quality, making starch a key determinant of grain yield and quality.

[0003] AP2 / ERF transcription factors are a class of plant transcriptional regulators with diverse biological functions. Previous research on AP2 / ERF transcription factors has largely focused on plant responses to biotic and abiotic stresses. Recent studies have shown that the AP2 / ERF family is involved in regulating crop quality. In rice, Rsr1, a member of the AP2 / ERF family, regulates starch structure and gelatinization temperature by repressing the expression of starch synthesis genes in the endosperm. Rsr1 mutants exhibit high amylose content, loose starch granules, and low gelatinization temperature, directly impacting edible quality. In barley, HvDREB1 can improve malt saccharification capacity by regulating the β-amylase gene.

[0004] The starch content in wheat grains plays a vital role in wheat yield and processing quality. However, most of the previous research by cultivators focused on improving and balancing wheat yield and quality through cultivation techniques such as water and fertilizer application. There is currently little research on the molecular mechanisms that coordinately regulate wheat yield and quality. Summary of the Invention

[0005] In order to solve the problem that there are few molecular mechanisms for the coordinated regulation of wheat yield and quality, the present invention provides a TaAP2-4 The application of genes in regulating the starch content of wheat grains, from the perspective of genetic engineering, fully explores the genes that positively regulate wheat quality and yield, which can not only provide genetic resources for gene aggregation breeding, but also is a new way to breed wheat quality.

[0006] In order to achieve the above object, the present invention provides a TaAP2-4 Application of a gene in regulating starch content in wheat grains, wherein the application is any one of the following: (1) Enhance the TaAP2-4 The expression of the gene increases the starch content of wheat grains; (2) Weaken the TaAP2-4 The expression of the gene reduces the starch content of wheat grains; described TaAP2-4 The gene is located on chromosome 4 of the wheat genome, and there are three copies on chromosome 4, named TaAP2-4-A, TaAP2-4-B and TaAP2-4-D respectively; the nucleotide sequence of TaAP2-4-A is shown in SEQ ID No. 10, the nucleotide sequence of TaAP2-4-B is shown in SEQ ID No. 11, and the nucleotide sequence of TaAP2-4-D is shown in SEQ ID No. 12.

[0007] Specifically, knocking out the TaAP2-4-A, TaAP2-4-B and TaAP2-4-D genes reduces the starch content of the grains in the wheat material.

[0008] The second aspect of the present invention provides the above TaAP2-4 A CRISPR / Cas9 vector for a gene, comprising an expression cassette TaU3-sgRNA1 for regulating the expression of sgRNA1 by the wheat TaU3 promoter, an expression cassette TaU3-sgRNA2 for regulating the expression of sgRNA2 by the wheat TaU3 promoter, and an expression cassette for regulating the expression of Cas9 by the promoter Ubi; The sgRNA expression cassette elements include the following elements from upstream to downstream: TaU3 promoter, TaAP2-4 The target sequence of sgRNA1, terminator OsU3t, TaU3 promoter, TaAP2-4 The target sequence is sgRNA2, and the terminator is TaU3t.

[0009] Specifically, the TaAP2-4 The sgRNA1 sequence of the gene is shown in SEQ ID No. 1; described TaAP2-4 The sgRNA2 sequence of the gene is shown in SEQ ID No. 2; The expression cassette TaU3-sgRNA1 sequence is specifically shown as SEQ ID No. 3; The expression cassette TaU3-sgRNA2 sequence is specifically shown in SEQ ID No. 4; The expression cassette sequence of Cas9 expression regulated by the promoter Ubi is specifically shown in SEQ ID No. 5; The expression cassette TaU3-sgRNA1 and the expression cassette TaU3-sgRNA2 are located upstream of the expression cassette of Cas9 expression regulated by the promoter Ubi, and the expression cassette TaU3-sgRNA1 and the expression cassette TaU3-sgRNA2 and the expression cassette of Cas9 expression regulated by the promoter Ubi are connected in series.

[0010] The third aspect of the present invention provides a transformant of the above-mentioned vector.

[0011] The fourth aspect of the present invention provides the above-mentioned vector in mutant wheat TaAP2-4 Application on.

[0012] A fifth aspect of the present invention provides a method for constructing the above-mentioned vector, which comprises the following steps: 1) Design targeting TaAP2-4 The sgRNA1 and sgRNA2 of the gene were connected to the 5' end of the primers used to amplify the OsU3t and TaU3 promoter backbone sequences to obtain primers TaAP2-4-gR1-F0 and TaAP2-4-gR2-R0, whose sequences are shown in SEQ ID No. 6 and SEQ ID No. 7; The primers were mixed to amplify the OsU3t and TaU3 promoter backbone sequences to obtain product sequences; 2) Using the TaAP2-4-gR1-F amplification primer set as SEQ ID No. 8 and the TaAP2-4-gR2-R amplification primer set as SEQ ID No. 9, the BsaI restriction site sequence in the pBUE411 vector was ligated to the 5' end of the PCR product obtained in step 1). After mixing the primers, the PCR product sequence obtained in step 1) was amplified to obtain the product sequence; 3) Perform gel recovery on the PCR product obtained in step 2) to obtain a single PCR fragment; 4) Ligate the recovered single PCR fragment with the pBUE411 vector using T4 ligase to obtain the final vector; 5) The ligation product obtained in step 4) was transformed into E. coli competent DH5α, spread on LB plates containing Kana antibiotics, screened, and single clones were selected. The bacterial solution was identified by PCR and Sanger sequencing using universal primers pBUE411-F and pBUE411-R to obtain TaAP2-4 CRISPR / Cas9 gene editing vector.

[0013] A sixth aspect of the present invention provides TaAP2-4 The transgenic knockout line material is obtained by extracting the plasmid from the correctly sequenced monoclonal clone obtained above and transferring it into Agrobacterium, and then genetically transforming the transformed Agrobacterium to obtain TaAP2-4 Transgenic knockout line materials.

[0014] Through the above technical solution, the present invention achieves the following beneficial effects: 1. The present invention successfully constructed a Crispr / Cas9 gene editing vector based on Crispr / Cas9 gene editing technology and achieved TaAP2-4 Gene frameshift mutation, to achieve TaAP2-4 Gene knockout purpose.

[0015] 2. The present invention has obtained TaAP2-4 Gene knockout wheat mutant strains with reduced starch content in grains TaAP2-4 The gene plays a positive regulatory role in the starch synthesis process in wheat grains.

[0016] 3. The present invention is based on TaAP2-4 The gene plays a positive regulatory role in the synthesis of starch in wheat grains and can be used TaAP2-4 Genes are used to create high-quality wheat varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 for TaAP2-4 Sequence alignment results of some homologous genes. The red lines mark the positions of gene knockout target sgRNA1 and sgRNA2. Figure 2 This is a gel image of the PCR product of the MT1T2 vector amplified using primers, where the product size is 964 bp; Figure 3 for TaAP2-4 The positive colony detection image of the gene knockout vector construction, the sizes of the positive colony PCR products are 1000bp and 1919bp; Figure 4 for TaAP2-4 Sequence variation pattern of gene knockout transgenic lines; Figure 5 for TaAP2-4 Grain length phenotype of gene knockout transgenic lines; Figure 6 for TaAP2-4 Grain width phenotype of gene knockout transgenic lines; Figure 7 for TaAP2-4 Determination of amylose content in grains of gene knockout transgenic lines; Figure 8 for TaAP2-4 Determination of amylopectin content in grains of gene knockout transgenic lines. DETAILED DESCRIPTION

[0018] The following is a detailed description of the specific embodiments of the present invention in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0019] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0020] The wheat materials used in the present invention are all germplasm resources materials preserved in the Yangzhou University Agricultural College-Jiangsu Provincial Germplasm Resources Bank (crops), and can be obtained and used by technicians and researchers in this field.

[0021] Example 1 TaAP2-4 Design of knockout target sgRNA1 and sgRNA2 TaAP2-4 The gene is located on chromosome 4 of the wheat genome. There are three copies of the gene on chromosome 4. The sequences of these three copies were amplified from the wheat variety Yangmai 18 and named TaAP2-4-A, TaAP2-4-B and TaAP2-4-D respectively. The amplified sequences are as follows Figure 1 As shown, the underlined part is the sgRNA1 and sgRNA2 sequences used to target and knock out these three copies, and the first three digits of the underlined part are the PAM sequence CCG.

[0022] Example 2 Construction TaAP2-4 Knockout transgenic vector 1) Design targeting TaAP2-4 The sgRNA1 and sgRNA2 of the gene were connected to the 5' end of the primers used to amplify the OsU3t and TaU3 promoter backbone sequences to obtain primers TaAP2-4-gR1-F0 and TaAP2-4-gR2-R0, whose sequences are shown in SEQ ID No. 6 and SEQ ID No. 7; The primers were mixed to amplify the OsU3t and TaU3 promoter backbone sequences, and the product sequence was obtained with a size of 931 bp; The primer mixture system was as follows: TaAP2-4-gR1-F0 and TaAAP2-4-gR1-R0 with a final concentration of 0.5 μmol / L; The PCR amplification system is as follows: 20-100 ng of MT1T2 plasmid containing the OsU3t terminator A1 and TaU3 promoter A2 sequences, 1 μL, primer mix 2 μL, 2 × Phanta™ Master Mix 10 μL, and H2O added to make up to 20 μL; The reaction conditions for PCR amplification were as follows: pre-denaturation at 95°C for 3 min, followed by denaturation at 95°C for 10 sec, annealing at 58°C for 30 sec, and extension at 72°C for 40 sec, for 35 cycles; and a final extension at 72°C for 5 min; and the samples were stored at 15°C.

[0023] 2) Design a pair of amplification primers to connect the BsaI restriction site sequence in the pBUE411 vector to the 5' end of the PCR product obtained in step 1). The amplification primers are TaAP2-4-gR1-F as shown in SEQ ID No. 8 and TaAP2-4-gR2-R as shown in SEQ ID No. 9; After the primers are mixed, the PCR product sequence obtained in step 1) is amplified to obtain a product sequence with a size of 964 bp.

[0024] The primer mixture system was as follows: the final concentration of TaAP2-4-gR1-F0 and TaAP2-4-gR1-R0 was 0.5 μM; the final concentration of TaAP2-4-gR1-F and TaAP2-4-gR1-R was 10 μM.

[0025] The PCR amplification system was as follows: 10 μL of the PCR product obtained in step 1, 4 μL of the primer mixture, 50 μL of 2 × Phanta™ Master Mix, and H O added to make up to 100 μL; The reaction conditions for PCR amplification were as follows: pre-denaturation at 95°C for 3 min, followed by denaturation at 95°C for 10 sec, annealing at 58°C for 30 sec, and extension at 72°C for 40 sec, for 35 cycles; and a final extension at 72°C for 5 min; and the samples were stored at 15°C.

[0026] 3) The PCR product obtained in step 2) was subjected to gel recovery experiment to obtain a single PCR fragment.

[0027] After electrophoresis of 100 μL of PCR product obtained in step 2), the agarose gel containing the target band was cut out and placed in a clean centrifuge tube and weighed. Then, an appropriate amount of PN solution was added to dissolve the gel block. The amount of PN added was calculated based on the weight of the gel block. 100 μL of PN was added for every 0.1 g of gel block. After the gel block was completely dissolved, the liquid was transferred to an adsorption column, allowed to stand at room temperature for 2 minutes, centrifuged at 12,000 rpm for 1 minute, the waste liquid was discarded and the centrifugation step was repeated once. 600 μL of PW solution was added to the adsorption column, centrifuged at 12,000 rpm for 1 minute, the waste liquid was discarded and the centrifugation step was repeated once. After discarding the waste liquid, the empty tube was centrifuged at 12,000 rpm for 2 minutes, and the adsorption column was air-dried at room temperature for 10 minutes. The air-dried adsorption column was placed in a centrifuge tube and 30 μL of water was added. After standing at room temperature for two minutes, it was placed in a centrifuge and centrifuged at 12,000 rpm for 2 minutes to obtain a single PCR fragment.

[0028] The gel recovery kit is the Tiangen company's agarose gel DNA recovery kit (enhanced).

[0029] 4) Ligate the recovered single PCR fragment with the pBUE411 vector using T4 ligase to obtain the final vector.

[0030] Mix 2 μL of the PCR product obtained in step 3), 1 μL of the restriction endonuclease BsaI, 1.5 μL of the restriction endonuclease buffer (CutSmart Buffer), 2 μL of the pBUE414 vector plasmid, 1 μL of T4 ligase, and 1 μL of 10xNEB T4 Buffer. Add water to make up to 15 μL. Incubate the vector enzyme digestion and ligation reaction in a 37°C water bath for 5 hours. Inactivate the reaction in a PCR machine at 50°C for 5 minutes and then at 80°C for 10 minutes to complete the Crispr / Cas9 vector ligation of the TaAP2-4 gene.

[0031] 5) Transformation verification of the Crispr / Cas9 vector obtained in step 4 The ligation product obtained in step 4) was transformed into competent E. coli DH5α and plated on LB plates containing Kana antibiotics for screening. Single clones were selected for bacterial liquid PCR identification using universal primers pBUE411-F and pBUE411-R. PCR products were run on agarose gels. Positive clones were identified as having bands of 1919 bp and 1000 bp (refer to Figure 3 The bacterial culture was sent to Yangzhou Lanke Biotechnology Co., Ltd. for sequencing.

[0032] 6) The clones that were sequenced correctly were re-propagated, the plasmids were extracted, and the clones were transformed into GV3101 competent Agrobacterium. The positive clones were selected and sent to the Shandong Academy of Agricultural Sciences for genetic transformation. The recipient was the wheat line Yangmai 18.

[0033] Example 3 Identification of TaAP2-4 knockout transgenic lines After obtaining T0 generation transgenic plants from the Shandong Institute of Agricultural Sciences, the seedlings grew and developed well, and leaves were collected to extract DNA using the CTAB method. The extracted DNA was then used as a template and 411 F and 411 R as primers to detect whether the CRISPR / Cas9 gene editing vector had been successfully transformed. The plants that were successfully transformed were subjected to additional generation treatment.

[0034] TaAP2-4 Whether the gene knockout is successful requires deep sequencing to determine. The detection fragment is amplified using the above-extracted DNA as a template and the specific primers TaAP2-4-AF, TaAP2-4-AR, TaAP2-4-BF, TaAP2-4-BR, TaAP2-4-DF, and TaAP2-4-DR on both sides of the target sequence. The amplified product is subjected to Sanger sequencing. The results are as follows Figure 5 shown.

[0035] The sequence of TaAP2-4-AF is shown in SEQ ID NO. 13; The TaAP2-4-AR sequence is shown in SEQ ID NO. 14; The sequence of TaAP2-4-BF is shown in SEQ ID NO. 15; The TaAP2-4-BR sequence is shown in SEQ ID NO. 16; The sequence of TaAP2-4-DF is shown in SEQ ID NO. 17; The TaAP2-4-DR sequence is shown in SEQ ID NO. 18; Figure 4 In the figure, "TaAP2-4-target1" indicates target sequence 1, "TaAP2-4-target2" indicates target sequence 2, "TaAP2-4-A", "TaAP2-4-B", and "TaAP2-4-D" indicate the sequences of the TaAP2-4 gene at the target sites on chromosomes 4A, 4B, and 4D in the wild-type Yangmai 18 wheat variety; KO-7 and KO-10 are pure lines with TaAP2-4 knockout mutations; comparison Figure 4 The results showed that in KO-7, TaAP2-4-A deleted 50 bases starting from the 94th nucleotide position and inserted the nucleotide T at the 144th nucleotide position, TaAP2-4-B deleted the nucleotides at positions 91 to 140, TaAP2-4-D deleted 53 bases after the 91st nucleotide position and inserted the nucleotide T at the 144th nucleotide position, resulting in simultaneous frameshift mutations in the three TaAP2-4 partial homologous genes and premature termination of their protein translation. Similarly, in KO-10, TaAP2-4-A deleted 53 bases starting from the 94th nucleotide position, TaAP2-4-B deleted 49 bases starting from the 92nd nucleotide position, TaAP2-4-D deleted the 92nd to 145th nucleotides and inserted the nucleotide T at the 145th nucleotide position, resulting in simultaneous frameshift mutations in the three TaAP2-4 partial homologous genes and premature termination of their protein translation (refer to Figure 4 Frameshift mutations occurred in all three copies of chromosomes 4A, 4B, and 4D in both KO-7 and KO-10 lines, demonstrating successful knockout of the TaAP2-4 gene.

[0036] Example 4 TaAP2-4 Application of genes in increasing starch content in wheat grains like Figure 6 As shown, TaAP2-4 The grain length and width of the two knockout mutant lines KO-7 and KO-10 were smaller than those of the wild type Yangmai 18 (refer to Figure 5 、 Figure 6). Wheat grain starch content is an important factor affecting the formation of wheat grain size. In order to study TaAP2-4 To investigate whether the gene affects starch synthesis in wheat grains, the present invention tested the starch content of wild-type Yangmai 18 and mutants KO-7 and KO-10. We used a dual-wavelength method to measure the starch content in wild-type and mutant grains. The results showed that the amylose content and amylopectin content of mutants KO-7 and KO-10 were significantly lower than those of wild-type Yangmai 18 (refer to Figure 7 / Figure 8 ). This indicates TaAP2-4 The knockout of affected the synthesis and accumulation of starch in wheat grains.

[0037] The specific steps for determining the starch content of wheat grains using the dual-wavelength method are as follows: (1) Experimental reagents: anhydrous ethanol; 0.5 mol / L potassium hydroxide solution (weigh 2.8 g KOH and dissolve it in distilled water to a final volume of 100 mL); 0.1 mol / L HCl solution (weigh 8.4 mL concentrated hydrochloric acid and dilute it with distilled water to a final volume of 1000 mL); iodine reagent (weigh 2.0 g potassium iodide, dissolve it in a small amount of distilled water, add 0.2 g iodine, and finally dilute it with distilled water to a final volume of 100 mL); ether; standard samples of amylose and amylopectin.

[0038] (2) Experimental steps: ① Draw a dual-wavelength amylose standard curve: Dissolve 0.1 g of pure amylose in 10 mL of 0.5 mol / L KOH solution in a 10 mL volumetric flask. Once completely dissolved in a hot water bath, dilute the solution to 100 mL with distilled water in a 100 mL volumetric flask. Mix thoroughly to obtain a 1 mg / mL amylose standard solution. Place 0.3, 0.5, 0.7, 0.9, 1.1, and 1.3 mL of each standard solution into a 50 mL volumetric flask. Add 20-30 mL of distilled water to dilute the solution. Adjust the pH to approximately 3.5 with 0.1 mol / L hydrochloric acid solution, add 0.5 mL of iodine reagent, and finally dilute the solution to 50 mL with distilled water. Mix thoroughly and let stand for 20 minutes. Using distilled water as a blank control, measure the optical density at 480 nm and 554 nm using a microplate reader. Plot the dual-wavelength amylose standard curve using the difference between the two optical densities as the y-axis and the amylose concentration as the x-axis.

[0039] ② Draw a dual-wavelength amylopectin standard curve: The first part of the steps is basically the same as the steps for drawing a dual-wavelength amylose standard curve. Subsequently, the optical density needs to be measured at wavelengths of 631 nm and 754 nm, and the dual-wavelength amylopectin standard curve needs to be drawn with the difference between the two optical densities as the vertical axis and the amylopectin concentration as the horizontal axis.

[0040] ③ Determination of amylose, amylopectin, and total starch content in the sample: Grind the mature kernels using a sample grinder, pass through a 60-mesh sieve, and defatted with sample-soaked ether. Weigh 0.1 g of the defatted sample and dissolve it in 10 mL of 0.5 mol / L KOH solution. Boil the sample in boiling water for 10 minutes to fully dissolve it. Remove the sample and dilute it to 50 mL with distilled water in a volumetric flask. After allowing it to stand, two 2.5 mL aliquots of the sample solution were drawn up as the sample assay solution and the sample blank solution. After dilution with 20-30 mL of distilled water, the pH was adjusted to approximately 3.5 with 0.1 mol / L hydrochloric acid solution. 0.5 mL of iodine reagent was added, and the sample was diluted to 50 mL with distilled water. Mix thoroughly and allow to stand for 20 minutes. Using distilled water as a blank control, the optical density was measured at 480 nm, 554 nm, 631 nm, and 754 nm using a microplate reader. The amylose, amylopectin, and total starch contents of the sample were calculated based on the dual-wavelength curves of the two starches.

[0041] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0042] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0043] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A TaAP2-4 The application of a gene in regulating the starch content of wheat grains is characterized in that: The application is any of the following: (1) Enhance the TaAP2-4 The expression of the gene increases the starch content of wheat grains; (2) Weaken the TaAP2-4 The expression of the gene reduces the starch content of wheat grains; described TaAP2-4 The gene is located on chromosome 4 of the wheat genome, and there are three copies on chromosome 4, namely TaAP2-4-A, TaAP2-4-B and TaAP2-4-D; the nucleotide sequence of TaAP2-4-A is shown in SEQ ID No. 10, the nucleotide sequence of TaAP2-4-B is shown in SEQ ID No. 11, and the nucleotide sequence of TaAP2-4-D is shown in SEQ ID No.

12.

2. The use according to claim 1, characterized in that Knocking out the TaAP2-4-A, TaAP2-4-B and TaAP2-4-D genes reduces the starch content of grains in wheat materials.

3. The method according to claim 1 TaAP2-4 A CRISPR / Cas9 vector for a gene, characterized in that Including the expression frame TaU3-sgRNA1 regulated by the wheat TaU3 promoter, the expression frame TaU3-sgRNA2 regulated by the wheat TaU3 promoter, and the expression frame Ubi promoter regulated the expression of Cas9; The sgRNA expression cassette elements include the following elements from upstream to downstream: TaU3 promoter, TaAP2-4 The target sequence of sgRNA1, terminator OsU3t, TaU3 promoter, TaAP2-4 The target sequence is sgRNA2, and the terminator is TaU3t.

4. The carrier according to claim 3, characterized in that described TaAP2-4 The sgRNA1 sequence of the gene is shown in SEQ ID No. 1; described TaAP2-4 The sgRNA2 sequence of the gene is shown in SEQ ID No. 2; The expression cassette TaU3-sgRNA1 sequence is shown in SEQ ID No. 3; The expression cassette TaU3-sgRNA2 sequence is shown in SEQ ID No. 4; The expression cassette sequence of Cas9 expression regulated by the promoter Ubi is shown in SEQ ID No. 5; The expression cassette TaU3-sgRNA1 and the expression cassette TaU3-sgRNA2 are located upstream of the expression cassette of Cas9 expression regulated by the promoter Ubi, and the expression cassette TaU3-sgRNA1 and the expression cassette TaU3-sgRNA2 and the expression cassette of Cas9 expression regulated by the promoter Ubi are connected in series.

5. A transformant containing the vector according to claim 3 or 4.

6. The vector according to claim 3 or 4 is used to express mutant wheat TaAP2-4 Application on.

7. The method for constructing the vector according to claim 3 or 4, characterized in that: The steps include: 1) Design targeting TaAP2-4 The sgRNA1 and sgRNA2 of the gene were connected to the 5' end of the primers used to amplify the OsU3t and TaU3 promoter backbone sequences to obtain primers TaAP2-4-gR1-F0 and TaAP2-4-gR2-R0, whose sequences are shown in SEQ ID No. 6 and SEQ ID No. 7; The primers were mixed to amplify the OsU3t and TaU3 promoter backbone sequences to obtain product sequences; 2) Using the TaAP2-4-gR1-F amplification primer set as SEQ ID No. 8 and the TaAP2-4-gR2-R amplification primer set as SEQ ID No. 9, the BsaI restriction site sequence in the pBUE411 vector was ligated to the 5' end of the PCR product obtained in step 1). After mixing the primers, the PCR product sequence obtained in step 1) was amplified to obtain the product sequence; 3) Perform gel recovery on the PCR product obtained in step 2) to obtain a single PCR fragment; 4) Ligate the recovered single PCR fragment with the pBUE411 vector using T4 ligase to obtain the final vector; 5) The ligation product obtained in step 4) was transformed into E. coli competent DH5α, spread on LB plates containing Kana antibiotics, screened, and single clones were selected. The bacterial solution was identified by PCR and Sanger sequencing using universal primers pBUE411-F and pBUE411-R to obtain TaAP2-4 CRISPR / Cas9 gene editing vector.

8. TaAP2-4 Transgenic knockout material, characterized in that The material acquisition process is as follows: extract the plasmid of the monoclonal clone with correct sequencing obtained in claim 7 and transfer it into Agrobacterium, then genetically transform the transformed Agrobacterium, and finally obtain TaAP2-4 Transgenic knockout line materials.

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