Application of TaCSD gene and protein thereof in wheat genetic transformation

Abstract

The invention discloses an application of a TaCSD gene and a protein thereof in wheat genetic transformation, and belongs to the technical field of plant genetic engineering. The invention finds that the over-expression of the TaCSD gene can significantly improve the genetic transformation efficiency of wheat for the first time. By utilizing the TaCSD gene, the conversion efficiency of nucleic acid molecules integrated into a wheat genome can be improved, genetic improvement of wheat varieties is facilitated, and the TaCSD gene has important significance on production and cultivation of wheat.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to the application of a TaCSD gene and a protein thereof in wheat genetic transformation. Background Art

[0002] As an important global food crop, the genetic improvement of wheat (Triticum aestivum L.) is of great significance to ensuring food security. However, wheat genetic transformation has long faced problems such as low efficiency and strong genotype dependence, which seriously restricts the research of gene function and the progress of biological breeding. Genetic transformation is a core tool of modern biotechnology, which can introduce exogenous genes into the plant genome to achieve precise improvement of target traits. The efficient regeneration of wheat plants is the basis of genetic transformation. The current wheat in vitro regeneration system has a certain degree of instability: callus tissue is susceptible to oxidative stress damage during long-term tissue culture, resulting in loss of embryonic ability and decreased regeneration efficiency.

[0003] Plant regeneration in vitro relies on the reprogramming of somatic cells into embryonic cells, and reactive oxygen species (ROS) exhibit dual functions in this process. Studies have shown that low concentrations of ROS can activate cell proliferation signals, while excessive accumulation can trigger lipid peroxidation, cell cycle arrest, and apoptosis (Bhattacharjee, 2005). Abnormal accumulation of ROS (such as H₂O₂ and O₂⁻) during the induction and differentiation stages of plant callus may be a key factor contributing to plant regeneration failure (Zhao et al., 2021).

[0004] Superoxide dismutase (SOD), a core component of the ROS scavenging system, maintains intracellular redox homeostasis by catalyzing the conversion of O₂- to H₂O₂ and O₂. Among these, copper-zinc-type SOD (CSD) is localized in the cytoplasm and chloroplasts, and its expression level is directly correlated with the antioxidant capacity of plants (Alscher et al., 2002). Currently, research on CSD genes primarily focuses on plant stress tolerance. Overexpression of the CuZnSOD gene in sweet potato effectively protects against salt stress (Hui Yan et al., 2016). Overexpression of the StSOD1 gene enhances potato tolerance to low temperatures (Che et al., 2020). Although the functions of CSD genes in plants have been preliminarily elucidated, their role in regulating wheat regeneration remains understudied.

[0005] In recent years, the discovery and functional analysis of regeneration-related transcription factors have provided a new direction for breaking through wheat genotype dependence. Overexpressing these regeneration genes can effectively improve wheat regeneration capacity and genetic transformation efficiency. However, the number of regeneration-related genes isolated to date is still very small. Therefore, further isolation and identification of key genes that promote wheat regeneration is necessary, which is of great practical significance for improving crop agronomic traits and promoting biotechnology breeding. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide an application of a TaCSD gene and its protein in wheat genetic transformation.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect of the present invention, a use of the TaCSD gene in improving the efficiency of wheat genetic transformation is provided; the TaCSD gene is a nucleic acid molecule as shown in the following i) or ii) or iii):

[0009] i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1;

[0010] ii) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO. 2 other than i);

[0011] iii) a DNA molecule that has 90% or more identity with the DNA fragment defined in i) or ii), and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO. 2.

[0012] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. Identity can be evaluated using computer software, for example, the BLAST algorithm (Altschul et al. 1990. Journal of Molecular Biology 215: 403-410; Karlin and Altschul. 1993. Proceedings of the National Academy of Sciences 90: 5873-5877).

[0013] In the above nucleic acid molecules, the 90% or greater homology may be at least 90%, 92%, 93%, 95%, 96%, 98% or 99% identity.

[0014] In the above application, the genetic transformation efficiency of wheat can be improved by overexpressing the TaCSD gene.

[0015] In the above application, the genetic transformation efficiency refers to the ratio of the number of positive seedlings obtained after genetic manipulation of wheat immature embryos to the total number of immature embryos.

[0016] The second aspect of the present invention provides the use of TaCSD protein in improving the efficiency of wheat genetic transformation;

[0017] Preferably, the TaCSD protein is the protein shown in (A1) or (A2) below:

[0018] (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO. 2 in the sequence listing;

[0019] (A2) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0020] In the above proteins, a protein tag refers to a polypeptide or protein that is fused and expressed with a target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. To facilitate purification of the protein in (A1), a tag may be attached to the amino or carboxyl terminus of the protein in (A1). The tag may be Poly-Arg (usually six RRRRRs), Poly-His (usually six HHHHHHs), FLAG (DYKDDDDK), Strep-tag II (WSHPQFEK), or c-Myc (EQKLISEEDL).

[0021] The third aspect of the present invention provides the use of an expression cassette, a recombinant expression vector or a recombinant bacterium containing the TaCSD gene in improving the genetic transformation efficiency of wheat.

[0022] In the above application, the recombinant expression vector can be constructed using an existing plant expression vector. Preferably, the pUbi110 expression vector is used to construct the recombinant expression vector containing the TaCSD gene.

[0023] A fourth aspect of the present invention provides a method for improving wheat genetic transformation efficiency, comprising the step of overexpressing the TaCSD gene.

[0024] In the above method, the method of overexpressing the TaCSD gene includes: exogenously introducing the TaCSD gene through the polyethylene glycol method, the Agrobacterium-mediated method or the gene gun bombardment method.

[0025] Preferably, the TaCSD gene is exogenously introduced using the Agrobacterium-mediated method, specifically: the TaCSD gene is connected to an expression vector to construct a recombinant expression vector, the recombinant expression vector is transferred into Agrobacterium competent cells to obtain an Agrobacterium strain for transformation; and the Agrobacterium strain is used to infect wheat embryos.

[0026] Beneficial effects of the present invention:

[0027] This study, published in the journal Nature Communications, demonstrates that overexpressing the TaCSD gene in the wheat variety Yangmai 158 significantly improves the efficiency of genetic transformation. Utilizing the TaCSD gene can enhance the efficiency of nucleic acid integration into the wheat genome, facilitating the genetic improvement of wheat varieties and holding important implications for wheat production and cultivation. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the partial structure of the plant expression vector pUbi110-TaCSD.

[0029] Figure 2 Schematic diagram of the partial structure of the plant expression vector pUbi110-GUS.

[0030] Figure 3 The results of PCR-specific amplification of candidate transgenic plants obtained by infecting wheat with the plant expression vector pUbi110-TaCSD; 1-9 in the figure represent candidate transgenic plants, PC represents positive plasmid, NC represents negative control, WT represents wild-type plant, and M represents 2000 bp molecular weight marker.

[0031] Figure 4 The statistical results of callus induction rate of Yangmai 158 immature embryo explants transformed with plant expression vectors pUbi110-TaCSD and pUbi110-GUS are shown.

[0032] Figure 5 Statistical results of genetic transformation efficiency of Yangmai 158 immature embryo explants transformed with plant expression vectors pUbi110-TaCSD and pUbi110-GUS. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0034] As mentioned above, wheat genetic transformation technology has long faced challenges such as low efficiency and strong genotype dependence, severely hindering gene function research and bio-breeding progress. "Yangmai 158" is a first-generation, leading variety developed by the Lixiahe Agricultural Science Institute in Jiangsu Province using integrated breeding techniques based on the ecological characteristics of the middle and lower reaches of the Yangtze River. It has initially solved the challenge of achieving high yields on a large scale while maintaining resistance to ergot and powdery mildew in the warm and humid late-stage wheat growth zone. "Yangmai 158" has been on the market for many years and currently faces the challenge of further genetic improvement.

[0035] In light of this, the present invention conducted in-depth research on improving the genetic transformation efficiency of Yangmai 158 using the variety as the experimental subject. The present invention found that the TaCSD gene can be used as a regeneration gene to improve the genetic transformation efficiency of Yangmai 158. Overexpressing the TaCSD gene in Yangmai 158 significantly increased the callus induction rate and genetic transformation efficiency of Yangmai 158.

[0036] The nucleotide sequence of the TaCSD gene is shown in SEQ ID NO. 1, and is as follows:

[0037]

[0038] The amino acid sequence of the protein TaCSD encoded by the TaCSD gene is shown in SEQ ID NO. 2, and is as follows:

[0039] .

[0040] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0041] The unspecified test materials used in the examples of the present invention are all conventional test materials in this area and can be purchased through commercial channels. The present invention introduces the expression vector into the plant cell, and the introduction methods are all well known to those skilled in the art, including but not limited to: Agrobacterium-mediated method, gene gun bombardment method, electroporation method, ovary injection method, etc. The selective marker gene used in the present invention is the bar gene, which encodes the phosphinothricin acetyltransferase PAT protein. Other selective marker genes and reporter genes including nptII and hpt can be further used. The screening antibiotic selected by the present invention is phosphinothricin, and the selection of screening agents such as bialaphos can also have the same effect. Where specific experimental conditions and methods are not specified in the examples of the present invention, conventional conditions are generally used, such as J. Sambrook et al., ed., Science Press, 2002, Molecular Cloning Experiment Guide (3rd edition); DL Spector et al., ed., Science Press, 2001, Cell Experiment Guide; or according to the conditions recommended by the manufacturer.

[0042] Example 1: Cloning of TaCSD gene

[0043] 1. Test method:

[0044] Total RNA from wheat was extracted using Ultrapure RNA Kit (Chemical World, Cat. No. CW0581M). cDNA was reverse transcribed using the II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Nanjing Novozymes Biotechnology Co., Ltd., catalog number: R212).

[0045] PCR amplification was performed using the cDNA as a template using a primer pair (upstream primer: 5'-ATGGTGGTTCTTGCTGCTTC-3'; downstream primer: 5'-TTAGACTATTTGATAGTTCGCAGCAA-3'). The amplification system consisted of 2 μl of the upstream primer (10 μmol / μl), 2 μl of the downstream primer (10 μmol / μl), 12.5 μl of 2× Phanta Max Master Mix, 1 μl of the cDNA template, and ddH2O to a total volume of 25 μl. Amplification conditions were: initial denaturation at 95°C for 5 minutes, followed by 38 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 50 seconds, followed by extension at 72°C for 5 minutes.

[0046] The amplified PCR product was -Blunt3 Cloning Kit (Beijing Quanshijin Biotechnology Co., Ltd., Cat. No. CB301-01) operation steps connected to pEASY -Blunt3 vector to obtain pEASY-Blunt3-TaCSD vector, and sequenced it.

[0047] 2. Test results:

[0048] After sequencing analysis, the nucleotide sequence of the PCR amplification product is Sequence 1 in the sequence table, and the gene represented by the PCR product is named TaCSD gene; the protein encoded by the gene is named TaCSD, and the amino acid sequence of the protein is Sequence 2 in the sequence table.

[0049] Example 2: Construction of TaCSD gene overexpression vector and investigation of genetic transformation efficiency

[0050] 1. Test method:

[0051] (1) Construction of overexpression vector:

[0052] The single restriction endonuclease cleavage sites (SmaI, SpeI) on the overexpression vector pUbi110 were selected for vector enzymatic digestion, and the digestion products were recovered by gel recovery using a gel recovery / PCR product purification kit (Shandong Sikoje Biotechnology Co., Ltd., catalog number: AE0101-C).

[0053] Using the pEASY-Blunt3-TaCSD plasmid from Example 1 as a template, a homologous primer pair (upstream primer: 5'-CGACTCTAGAGGATCCCCGGGATGGTGGTTCTTGCTGCTTCC-3'; downstream primer: 5'-GAATTCCGGCTCGAGACTAGTTTAGACTATTTGATAGTTCGCAGCAA-3') was designed for PCR amplification. The amplification system consisted of 2 μl of upstream primer (10 μmol / μl), 2 μl of downstream primer (10 μmol / μl), 12.5 μl of 2× Phanta Max Master Mix, and 1 μl of cDNA template. The total volume was adjusted to 25 μl with ddH2O. Amplification conditions were: pre-denaturation at 95°C for 5 minutes, followed by 38 cycles of denaturation at 95°C for 30 seconds, annealing at 59°C for 30 seconds, and extension at 72°C for 50 seconds, and finally extension at 72°C for 5 minutes.

[0054] The amplified PCR product was measured with reference to 2×Ezmax Ultra Universal Clone Mix (Shanghai Tolo Biotechnology Co., Ltd.) was used for homologous recombination, and the enzyme digestion product of the pUbi110 vector was ligated and sequenced. The single clones with correct sequencing were extracted with the SPARKeasy Plasmid Mini-Scale Rapid Extraction Kit (Shandong SIKEJIE Biotechnology Co., Ltd., Cat. No. AD0101-C) to construct the TaCSD gene overexpression vector pUbi110-TaCSD. The schematic diagram of the vector structure is shown in the figure. Figure 1 shown.

[0055] pUbi110-GUS was used as a control vector. The construction method of the control vector is as follows:

[0056] Refer to NCBI ( https: / / www.ncbi.nlm.nih.gov / PCR amplification was performed using nucleotides 15108-16919 of Sequence ID: MN266288.1 on the website. The primer pair (upstream primer: 5'-ATGTTACGTCCTGTAGAA-3'; downstream primer: 5'-TCATTGTTTGCCTCCCTG-3') was used. The amplification system consisted of 2 μl of upstream primer (10 μmol / μl), 2 μl of downstream primer (10 μmol / μl), 12.5 μl of 2× Phanta Max Master Mix, 1 μl of cDNA template, and ddH2O added to bring the total volume to 25 μl. Amplification conditions were: initial denaturation at 95°C for 5 minutes, followed by 38 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 1 minute, followed by extension at 72°C for 5 minutes.

[0057] The amplified PCR product was The PCR product was ligated using the Blunt3 Cloning Kit (Cat. No. CB301-01, Beijing Quanshijin Biotechnology Co., Ltd.) to obtain pEASY-Blunt3-GUS, which was then sequenced. Sequencing analysis revealed that the gene expressed by the PCR product was named GUS.

[0058] Using pEASY-Blunt3-GUS as a template, a primer pair (upstream primer: 5'-CGACTCTAGAGGATCCCCGGGATGTTACGTCCTGTAGAAACCCCA-3'; downstream primer: 5'-GAATTCCGGCTCGAGACTAGTTTGTTTGCCTCCCTGCTGC-3') was designed for PCR amplification. The amplification system consisted of 2 μl of upstream primer (10 μmol / μl), 2 μl of downstream primer (10 μmol / μl), 12.5 μl of 2× Phanta Max Master Mix, 1 μl of cDNA template, and ddH2O to a total volume of 25 μl. Amplification conditions were: initial denaturation at 95°C for 5 minutes, followed by 38 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 1 minute, followed by extension at 72°C for 5 minutes.

[0059] The amplified PCR product was measured with reference to 2×Ezmax Ultra Universal Clone Mix (Shanghai Tolo Biotechnology Co., Ltd.) was used for homologous recombination, and the product of pUbi110 vector digestion was ligated and sequenced. The correct single clone was extracted with the SPARKeasy Plasmid Mini-Scale Rapid Extraction Kit (Shandong SPARKeasy Biotechnology Co., Ltd., Cat. No. AD0101-C) to obtain the pUbi110-GUS vector. The schematic diagram of the vector structure is shown below. Figure 2 shown.

[0060] (2) Preparation of recombinant Agrobacterium:

[0061] The pUbi110-TaCSD was transformed into Agrobacterium EHA105 competent cells, and an Agrobacterium strain suitable for transformation was obtained, which was named pUbi110-TaCSD / EHA105.

[0062] The pUbi110-GUS was transformed into Agrobacterium EHA105 competent cells, and an Agrobacterium strain suitable for transformation was obtained, which was named pUbi110-GUS / EHA105.

[0063] (3) Agrobacterium-mediated transformation of wheat immature embryos:

[0064] For detailed steps and methods for Agrobacterium-mediated transformation of wheat immature embryos, refer to Wheat (Triticum aestivum L.) Transformation Using Immature Embryos (Ishida et al., 2015). The basic steps for genetic transformation are as follows:

[0065] ① Three days before infection, inoculate pUbi110-TaCSD / EHA105 and pUbi110-GUS / EHA105 Agrobacterium onto YEP solid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, respectively, and incubate in the dark at 28°C for 2 days. Pick a single colony and inoculate it into YEP liquid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, and incubate it with shaking at 28°C and 220 rpm overnight. Pour the above Agrobacterium solution into a 2ml centrifuge tube, centrifuge at 6000 rpm for 5 minutes, discard the supernatant, and resuspend the pellet with resuspension buffer to obtain Agrobacterium resuspensions of pUbi110-TaCSD / EHA105 and pUbi110-GUS / EHA105, respectively.

[0066] ② The immature embryos of "Yangmai 158" about 14 days after flowering were collected and infected with Agrobacterium resuspensions of pUbi110-TaCSD / EHA105 and pUbi110-GUS / EHA105, respectively. The embryos were spread with the scutellum side facing up on WLS-AS medium (1 / 10MS basic medium, 1 / 10MS vitamins, 10 g / L glucose, 100 μM acetosyringone, 8 g / L agarose), and cultured in the dark in a 23°C incubator for 2 days.

[0067] ③ Transfer the co-cultured embryos to WLS-Res medium (MS minimal medium, MS vitamins, 2,4-D 0.5 mg / L, picloram 2.2 mg / L, glutamine 0.5 g / L, casein 0.1 g / L, MgCl2·6H2O 0.75 g / L, maltose 40 g / L, AgNO3 0.85 mg / L, vitamin C 100 mg / L, carbenicillin 250 mg / L, agarose 5 g / L) and culture in a dark incubator at 25°C for 5 days.

[0068] ④ Transfer the callus tissue after recovery culture to WLS-P5 medium (WLS-Res medium supplemented with PPT 5 mg / L) and culture in a 25°C incubator in the dark for 2 weeks.

[0069] ⑤The callus tissue was then transferred to WLS-P10 medium (WLS-Res medium supplemented with PPT 10 mg / L) and cultured in a 25°C incubator in the dark for 3 weeks.

[0070] ⑥ The callus was transferred to LSZ-P5 medium (MS basic medium, LS vitamins, zeatin 5 mg / L, sucrose 20 g / L, carbenicillin 250 mg / L, PPT 5 mg / L, phytagel 3 g / L) and cultured in a 25°C incubator under light for 2 weeks.

[0071] ⑦ Transfer the regenerated buds of wheat callus to LSF-P5 medium (MS basic medium, LS vitamins, IBA0.2 mg / L, sucrose 15 g / L, carbenicillin 250 mg / L, PPT 5 mg / L, phytogel 3 g / L) and culture in a 25°C incubator under light until the roots of the regenerated buds are about 1-2 cm long.

[0072] ⑧ Transplant the rooted seedlings into nutrient soil to obtain resistant seedlings of pUbi110-TaCSD and pUbi110-GUS respectively.

[0073] (4) PCR detection of candidate transgenic plants:

[0074] The CTAB method (Sambrook and Russell, Molecular Cloning Laboratory Manual, 2001) was used to extract genomic DNA from leaves of T0 generation wheat plants transfected with pUbi110-TaCSD and pUbi110-GUS vectors.

[0075] Specific primers were designed to identify the target gene (upstream primer: 5'-GCTCTAACCTTGAGTACCTA-3'; downstream primer: 5'-GCCAGTACCACTAATGTTCATGT-3'). The amplified sequence was 821 bp in length. The amplification system consisted of 1 μl of upstream primer (10 μmol / μl), 1 μl of downstream primer (10 μmol / μl), 10 μl of 2× Rapid Taq Master Mix, 1 μl of cDNA template, and ddH2O added to a total volume of 20 μl. Amplification conditions were: initial denaturation at 95°C for 5 minutes; 35 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 15 seconds; and extension at 72°C for 5 minutes. After PCR, the results were analyzed by 1% agarose gel electrophoresis.

[0076] (5) Statistics of wheat genetic transformation efficiency:

[0077] After wheat embryos were infected with Agrobacterium, callus formation was induced, and the number of embryos and callus formed was counted. After the callus was cultured on a screening medium, resistant calli were transferred to a differentiation medium. The resistant seedlings that differentiated were identified by PCR, and the number of positive seedlings was counted. The callus induction rate and genetic transformation efficiency were calculated respectively. The calculation formula is as follows:

[0078] Callus induction rate (%) = (number of calli formed ÷ total number of immature embryos) × 100%;

[0079] Genetic transformation efficiency (%) = (number of positive seedlings ÷ total number of immature embryos) × 100%.

[0080] 2. Test results:

[0081] PCR identification results Figure 3 As shown, the results show that transgenic lines 1, 2, and 4-9 contain an 821 bp target gene fragment, while the negative control and wild-type wheat do not contain the target fragment. The results show that this example successfully obtained plants overexpressing the TaCSD gene.

[0082] Immature embryos of Yangmai 158 were collected as explants and transformed with the pUbi110-TaCSD vector and the control vector pUbi110-GUS by Agrobacterium-mediated method. The statistical data showed that the callus induction rate of the control vector was 80.47%, while that of the pUbi110-TaCSD vector was increased to 89.81% ( Figure 4 In terms of genetic transformation efficiency, the control vector pUbi110-GUS was only 3.91%, while the pUbi110-TaCSD vector increased to 30.56% ( Figure 5 These results indicate that overexpressing the TaCSD gene significantly increases callus induction and genetic transformation efficiency in wheat. The application of this gene has important economic and social benefits for analyzing plant gene function and improving crop agronomic traits.

[0083] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. Application of the TaCSD gene in improving wheat genetic transformation efficiency; the TaCSD gene is a nucleic acid molecule as shown in the following i) or ii) or iii): i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO. 2 other than i); iii) a DNA molecule that has 90% or more identity with the DNA fragment defined in i) or ii), and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO.

2.

2. Application of TaCSD protein in improving wheat genetic transformation efficiency.

3. The use according to claim 2, characterized in that The TaCSD protein is the protein shown below (A1) or (A2): (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO. 2 in the sequence listing; (A2) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

4. Application of expression cassettes, recombinant expression vectors or recombinant bacteria containing the TaCSD gene in improving the efficiency of wheat genetic transformation.

5. The use according to claim 4, characterized in that The pUbi110 expression vector was used to construct a recombinant expression vector containing the TaCSD gene.

6. A method for improving wheat genetic transformation efficiency, characterized in that: The method includes the step of overexpressing the TaCSD gene.

7. The method according to claim 6, characterized in that The method of overexpressing the TaCSD gene includes: exogenously transferring the TaCSD gene through the polyethylene glycol method, the Agrobacterium-mediated method or the gene gun bombardment method.

8. The method according to claim 7, characterized in that The TaCSD gene was exogenously introduced by Agrobacterium-mediated method, specifically: the TaCSD gene was connected to an expression vector to construct a recombinant expression vector, the recombinant expression vector was transferred into Agrobacterium competent cells to obtain an Agrobacterium strain for transformation; and the Agrobacterium strain was used to infect wheat immature embryos.

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