Application of TaLOGL9 or TaARF11 gene in improvement of wheat genetic transformation efficiency

Abstract

The invention relates to the technical field of agriculture. Relates to an application of a TaLOGL9 or TaARF11 gene in improving the genetic transformation efficiency of wheat, an application of a protein coded by the TaLOGL9 or TaARF11 gene in improving the transformation efficiency of nucleic acid molecules integrated into a wheat genome, and an expression cassette of the TaLOGL9 or TaARF11 gene. The invention relates to an application of a recombinant expression vector or a recombinant bacterium in improving the conversion efficiency of nucleic acid molecules integrated into a wheat genome and a genetic transformation method for improving the wheat conversion efficiency. According to the present invention, the influence of the wheat gene TaLOGL9 or TaARF11 overexpression on the genetic transformation efficiency is tested; the disclosed TaGRF4-GIF1 and a skeleton vector without the wheat gene are used as a contrast, and the gene for promoting the genetic transformation efficiency of the wheat is found by transforming an overexpressed binary vector through the wheat. The genetic transformation efficiency of the transformation vector containing the TaLOGL9 or TaARF11 gene is improved to different degrees, an important gene resource is provided for genetic transformation of wheat, and a new choice is provided for biological breeding of wheat.

Description

Technical Field

[0001] The present invention relates to the field of agricultural technology, and specifically to application of TaLOGL9 or TaARF11 gene in improving wheat genetic transformation efficiency. Background Art

[0002] Genetically modified technology has become one of the important methods for plant genetic improvement. Genetically modified plants have been widely used around the world. For example, the United States, Brazil, Argentina and other countries have planted a large number of genetically modified soybeans, corn, cotton, etc. According to data from global consulting firm AgbioInvestor, in 2023, the global area of ​​genetically modified crops will increase by 1.9% over the previous year to 206.3 million hectares (3.09 billion mu), setting a record high. my country has also made important progress in the research and application of genetically modified plants. For example, the planting area of ​​genetically modified insect-resistant cotton has continued to expand, and genetically modified soybeans and corn have been issued with a number of safety certificates in China. At the same time, gene editing technology is also gradually expanding its application. For example, China has issued a number of gene editing safety certificates for soybeans, corn, rice and wheat.

[0003] Wheat is closely linked to food security, social and economic stability, and human nutrition and health with its high-yield, high-quality, stable and efficient production characteristics. However, as an allohexaploid plant, wheat has a large genome, rich repetitive sequences, and poor regeneration ability, which makes genetic transformation face great challenges. Agrobacterium-mediated transformation has become one of the commonly used methods in wheat transformation due to its low cost, simple operation, high efficiency of foreign gene introduction, low copy number, and good genetic stability.

[0004] Overexpression of key genes that promote regeneration can effectively improve crop regeneration ability and genetic transformation efficiency. This new method is gradually gaining wide recognition. However, the number of regeneration-related genes isolated so far is still limited. Therefore, further isolation and identification of key genes that promote wheat regeneration is of great practical significance for improving crop agronomic traits and promoting biotechnology breeding. Summary of the invention

[0005] The purpose of the present invention is to provide the application of TaLOGL9 or TaARF11 gene in improving the efficiency of wheat genetic transformation in view of the above problems. The genetic transformation efficiency of the vector transformation vector containing TaLOGL9 or TaARF11 gene is improved to varying degrees, providing important gene resources for wheat genetic transformation.

[0006] To achieve the above object, the present invention discloses: The use of TaLOGL9 or TaARF11 gene in improving wheat genetic transformation efficiency is characterized in that the nucleotide sequence of the TaLOGL9 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the TaARF11 gene is shown as SEQ ID NO.3.

[0007] The use of the protein encoded by the TaLOGL9 or TaARF11 gene in improving the transformation efficiency of nucleic acid molecules integrated into the wheat genome is characterized in that the amino acid sequence of the protein encoded by the TaLOGL9 gene is shown in SEQ ID NO.2, and the amino acid sequence of the protein encoded by the TaARF11 gene is shown in SEQ ID NO.4.

[0008] The invention relates to an application of an expression cassette, a recombinant expression vector or a recombinant bacterium containing a TaLOGL9 or TaARF11 gene in improving the conversion efficiency of nucleic acid molecules integrated into a wheat genome, wherein the nucleotide sequence of the TaLOGL9 gene is as shown in SEQ ID NO.1, and the nucleotide sequence of the TaARF11 gene is as shown in SEQ ID NO.3.

[0009] A genetic transformation method for improving wheat transformation efficiency, the method comprising the following steps: Step S1, obtaining and amplifying a gene fragment of TaLOGL9 or TaARF11, wherein the nucleotide sequence of the TaLOGL9 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the TaARF11 gene is shown in SEQ ID NO.3; Step S2, integrating the gene fragment of TaLOGL9 or TaARF11 into the wheat transformation vector; Step S3: wheat genetic transformation.

[0010] Preferably, step S1 comprises the following steps: Step S1.1, cloning the target gene; Step S1.2, recovering the target gene.

[0011] Preferably, step S1.1 comprises the following steps: Step S1.1.1: Amplify the target fragment using a high-fidelity enzyme. The amplification system includes: PrimeSTARMaxPremix 14-16μl; Primer F 0.8-1.2 μl; Primer R 0.8-1.2 μl; Target gene 0.8-1.2 μl; Add ddH2O to 30μl; The PCR amplification reaction program was as follows: pre-denaturation at 96-100°C for 2-4 min, denaturation at 96-100°C for 8-12 s, annealing at 55-65°C for 8-12 s, extension at 65-75°C for 25-35 s, for a total of 30-40 cycles, post-extension at 70-74°C for 8-12 min, and storage at 14-18°C; Step S1.1.2: After the PCR amplification is completed, agarose gel electrophoresis is performed to cut out the band of a preset size and recover and purify it; Step S1.2 includes the following steps: Step S1.2.1, use a gel detector to cut the target band and place it in a 1.5 ml centrifuge tube, add 250-350 μl of Binding Buffer, heat the centrifuge tube in a 50-60°C metal bath, and shake the centrifuge tube every 4-6 minutes to completely dissolve the gel; Step S1.2.2, after the solution is cooled to room temperature, transfer the solution to an adsorption column, let it stand for 2-4 min, centrifuge at 11000-13000 rpm for 0.5-1.5 min at room temperature, discard the waste liquid in the collection tube, add 250-350 μl of Binding Buffer solution to the adsorption column, let it stand for 2-4 min, centrifuge at 11000-13000 rpm for 0.5-1.5 min, and discard the waste liquid; Step S1.2.3, add 600-800ul of Wash Buffer solution to the adsorption column, centrifuge at 12000-14000rpm for 0.5-1.5min, and discard the waste liquid; Step S1.2.4, repeat step S1.2.3; Step S1.2.5, centrifuge at 12000-14000 rpm for 2-4 min, transfer the adsorption column to another new 1.5 ml centrifuge tube, open the cover of the adsorption column, and let it stand to evaporate the residual Wash Buffer solution; Step S1.2.6, add 25-35 μl of elution buffer to the adsorption column, let stand for 1-3 min, centrifuge at 12000-14000 rpm for 0.5-1.5 min, and store at -18--22°C for later use.

[0012] Preferably, step S2 comprises the following steps: Step S2.1, digesting the vector plasmid; Step S2.2, homologous recombination; Step S2.3, transformation of Escherichia coli DH5α; Step S2.4, Colony PCR Identification of Escherichia coli: Step S2.5, plasmid extraction; Step S2.6, plasmid sequencing; Step S2.7, transform Agrobacterium EHA105.

[0013] Preferably, in step S2.1, the enzyme digestion system comprises: 10×Fastdigestbuffer 2.5-3.5μl; SwaI 0.8-1.2 μl; Plasmid 14-16 μl; Add ddH2O to 30μl; Digest in a water bath at 36.5-37.5℃ for 1.5-2.5h, detect by agarose gel electrophoresis, cut out the 15.3kb band and recover; In step S2.2, homologous recombination is performed based on the recombination of the DNA fragment with the homologous sequence at the end of the linearized vector, and the insert fragment is cloned into the linearized vector. The connection system includes: SingleAssemblyCloningMix 4-6μl; Target fragment 80-120ng; Linearized vector 40-60ng; Add ddH2O to 10 μl; The reaction system was placed in a 45-55°C metal bath for 25-35 min, placed on ice for 2-4 min, and then transformed into E. coli DH5α.

[0014] Preferably, step S2.3 comprises the following steps: Step S2.3.1, place the competent E. coli in a -70--90°C refrigerator on ice for 3-5 minutes, then add the completed ligation system to the competent cell and place it in an ice bath for 25-35 minutes; Step S2.3.2, heat shock in 40-44°C water bath for 45-55 seconds, ice bath for 1.5-2.5 minutes; Step S2.3.3, add 400 μl of LB liquid culture medium into the clean bench, and culture on a shaker at 36.5-37.5°C for 45-55 min; Step S2.3.4, centrifuge at 4000-4400 rpm for 2-4 min, discard 320-360 μl of the supernatant, pipette and mix the remaining bacterial solution, spread it evenly on LB solid medium with Kan resistance, and culture it in an incubator at 36.5-37.5°C overnight; Step S2.4 includes the following steps: Step S2.4.1, pick a single colony from the culture medium and streak it on a new culture medium, and take a preset amount of colonies for PCR identification; Step S2.4.2: After PCR, perform agarose gel electrophoresis to identify, select positive colonies in LB liquid culture medium, culture them in a shaking incubator at 36.5-37.5°C overnight, and incubate the bacteria and extract the plasmid the next day; Step S2.5 includes the following steps: Step S2.5.1, collect the positive clones cultured overnight, centrifuge at 12800-13000 rpm for 2-4 min, and discard the supernatant; Step S2.5.2, add 220-280 μl of buffer P1 solution and vortex to fully suspend the bacteria; Step S2.5.3, add 220-280 μl of buffer P2 solution and mix slowly; Step S2.5.4, add 320-380 μl of buffer N3 solution and mix immediately; Step S2.5.5, centrifuge at 12800-13000 rpm for 8-12 min, aspirate the supernatant into the adsorption column, let stand for 1.5-2.5 min, centrifuge at 12800-13000 rpm for 0.5-1.5 min, and discard the waste liquid in the collection tube; Step S2.5.6, add 380-420 μl of Wash Buffer to the adsorption column, centrifuge at 12800-13000 rpm for 0.5-1.5 min, and discard the waste liquid; centrifuge at 12800-13000 rpm for 2-4 minutes to remove the residual Wash Buffer; Step S2.5.7, transfer the adsorption column into a new 1.5 ml tube, open the lid, let it stand for 4-6 minutes, and remove the remaining Wash Buffer; Step S2.5.8, add 40-60 μl of elution buffer, incubate at room temperature for two minutes, centrifuge at 12800-13200 rpm for 0.5-1.5 min; store at -18--22°C; Step S2.7 includes the following steps: Step S2.7.1, thaw the competent Agrobacterium on ice for 2-4 minutes, and add 1.5-2.5 μl of the correctly sequenced plasmid DNA; Step S2.7.2, ice bath for 4-6 min, liquid nitrogen freezing for 4-6 min, 36.5-37.5°C water bath for 4-6 min, stand on ice for 1.5-2.5 min, add 350-450 μl of YEP liquid culture medium in a sterile clean bench, and culture at 26-30°C with shaking for 1.5-2.5 hours; Step S2.7.3, centrifuge at 3800-4200 rpm for 1.5-2.5 min, discard 300-380 μl YEP liquid culture medium, evenly spread the remaining bacterial liquid on YEP solid culture medium with Kan resistance, and culture in an incubator at 26-30°C overnight.

[0015] Preferably, step S3 comprises the following steps: The wheat variety Fielder was used as the recipient, and the growth conditions were 15-17 h of light, 24-28 °C, 7-9 h of darkness, 18-20 °C, and topdressing was applied once during the tillering stage and the grain filling stage; Step S3.1, material disinfection and sterilization: peel the wheat kernels, disinfect them with 75% alcohol for 0.5-1.5 min, 10% sodium hypochlorite solution for 8-12 min, and wash them with sterile water 2-4 times; Step S3.2, embryo processing: peel off the embryos under a microscope and place them in the infection solution, wash them twice in the infection solution, add the infection solution again, and centrifuge at 3-5°C for 8-12 minutes; Step S3.3, bacterial solution preparation: pick a single colony and shake it overnight at 26-30°C, centrifuge it and discard the supernatant, then add the invasion dye solution to resuspend it; Step S3.4, infecting young embryos; aspirating the infection solution in the young embryo tube, adding the resuspended bacterial solution, and standing at room temperature for 4-6 minutes; Step S3.5, co-cultivation: embryos are transferred to co-cultivation medium, cultured in the dark at 21-25° C. for 1-3 days; Step S3.6, recovery culture: excise the embryonic axis, transfer to recovery medium, and culture in the dark at 23-27° C. for 4-6 days; Step S3.7, screening culture: transfer to screening medium 1, 23-27°C, dark culture for 1.5-2.5 weeks, then transfer to screening medium 2, 23-27°C, dark culture for 2-4 weeks; Step S3.8, differentiation culture: the material is transferred to differentiation medium and cultured at 23-27°C under light for 1.5-2.5 weeks; Step S3.9, rooting culture: the material is transferred to the rooting medium and cultured at 23-27° C. under light for 1.5-2.5 weeks.

[0016] In summary, the beneficial effects of the present invention are as follows: the present invention tests the effect of overexpression of wheat gene TaLOGL9 or TaARF11 on genetic transformation efficiency. Using the disclosed TaGRF4-GIF1 and the backbone vector without wheat gene as controls, the binary vector overexpressing wheat transformation was used to discover genes that promote wheat genetic transformation efficiency.

[0017] Using immature embryos of wheat variety Fielder as explants, the vector containing the target gene was introduced into the wheat genome by Agrobacterium-mediated method, and the resistant callus and regenerated transgenic plants were counted to obtain the transformation efficiency of different vectors. Compared with the transformation efficiency of the control backbone vector, the genetic transformation efficiency of the vector containing TaLOGL9 or TaARF11 gene was improved to varying degrees, providing important gene resources for wheat genetic transformation and new options for wheat biological breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of wheat genetic transformation and regeneration seedlings.

[0019] Figure 2 This is a schematic diagram of the wheat transformation vector p69.

[0020] Figure 3 This is a schematic diagram of the wheat transformation vector p52.

[0021] Figure 4 This is a schematic diagram of the wheat transformation vector p58.

[0022] Figure 5 This is a schematic diagram of the wheat transformation vector p59. DETAILED DESCRIPTION

[0023] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0024] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments: Example 1, application of TaLOGL9 or TaARF11 gene in improving wheat genetic transformation efficiency, the nucleotide sequence of TaLOGL9 gene is shown in SEQ ID NO.1, and the nucleotide sequence of TaARF11 gene is shown in SEQ ID NO.3.

[0025] Example 2, use of the protein encoded by TaLOGL9 or TaARF11 gene in improving the conversion efficiency of nucleic acid molecules into the wheat genome. The amino acid sequence of the protein encoded by the TaLOGL9 gene is shown in SEQ ID NO.2, and the amino acid sequence of the protein encoded by the TaARF11 gene is shown in SEQ ID NO.4.

[0026] Example 3, the use of an expression cassette, a recombinant expression vector or a recombinant bacterium of the TaLOGL9 or TaARF11 gene in improving the conversion efficiency of nucleic acid molecules into the wheat genome, the nucleotide sequence of the TaLOGL9 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the TaARF11 gene is shown in SEQ ID NO.3.

[0027] Example 4, a genetic transformation method for improving wheat transformation efficiency, comprising the following steps: Step S1, obtaining and amplifying a gene fragment of TaLOGL9 or TaARF11, the nucleotide sequence of the TaLOGL9 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the TaARF11 gene is shown in SEQ ID NO.3.

[0028] Specifically, based on the genome information of the wheat variety Chinese Spring, the sequence information of genes TaLOGL9 and TaARF11 was searched on the wheat genome website, primers were designed, and the corresponding gene fragments were amplified using the DNA of the wheat variety Chinese Spring as a template.

[0029] The sequence of primer TaLOGL9F is shown as SEQ ID NO.5.

[0030] The sequence of primer TaLOGL9R is shown in SEQ ID NO.6.

[0031] The sequence of primer TaARF11F is shown in SEQ ID NO.7.

[0032] The sequence of primer TaARF11R is shown in SEQ ID NO.8.

[0033] The TaGRF4-GIF1 gene fragment was amplified from the vector pGRF4-GIF1.

[0034] The sequence of primer TaGRF4F is shown in SEQ ID NO.9.

[0035] The sequence of primer TaGIF1R is shown as SEQ ID NO.10.

[0036] Step S1 includes the following steps.

[0037] Step S1.1, cloning the target gene.

[0038] Step S1.1 includes the following steps.

[0039] Step S1.1.1: Amplify the target fragment using a high-fidelity enzyme. The amplification system includes: PrimeSTARMaxPremix 14μl; Primer F 0.8 μl; Primer R 0.8 μl; Target gene 0.8 μl; Add ddH2O to 30μl; The PCR amplification reaction program was as follows: pre-denaturation at 96°C for 2 min, denaturation at 96°C for 8 s, annealing at 55°C for 8 s, extension at 65°C for 25 s, for a total of 30 cycles, post-extension at 70°C for 8 min, and storage at 14°C; Step S1.1.2: After the PCR amplification is completed, agarose gel electrophoresis is performed, and the band of a preset size is cut out and recovered and purified.

[0040] Step S1.2, recovering the target gene.

[0041] Step S1.2 includes the following steps: Step S1.2.1, use a gel detector to cut the target band and place it in a 1.5 ml centrifuge tube, add 250 μl of Binding Buffer, heat the centrifuge tube in a 50°C metal bath, and shake the centrifuge tube every 4 minutes to completely dissolve the gel; Step S1.2.2, after the solution is cooled to room temperature, transfer the solution to the adsorption column, let it stand for 2 min, centrifuge at 11000 rpm for 0.5 min at room temperature, discard the waste liquid in the collection tube, add 250 μl of Binding Buffer solution to the adsorption column, let it stand for 2 min, centrifuge at 11000 rpm for 0.5 min, and discard the waste liquid; Step S1.2.3, add 600ul of Wash Buffer solution to the adsorption column, centrifuge at 12000rpm for 0.5min, and discard the waste liquid; Step S1.2.4, repeat step S1.2.3; Step S1.2.5, centrifuge at 12000 rpm for 2 min, transfer the adsorption column to another new 1.5 ml centrifuge tube, open the cover of the adsorption column, and let it stand to evaporate the residual Wash Buffer solution; Step S1.2.6, add 25 μl of elution buffer to the adsorption column, let stand for 1 min, centrifuge at 12000 rpm for 0.5 min, and store at -18°C for later use.

[0042] Step S2, step S2, integrate the gene fragment of TaLOGL9 or TaARF11 into the wheat transformation vector. The p69 plasmid is cut with SwaI, and the plasmid is preserved in this laboratory, mainly including tobacco mosaic virus 35s promoter, glufosinate resistance gene bar, tobacco mosaic virus 35s polyA terminator, corn ubiquitin promoter, GUS gene, nos terminator, corn ubiquitin promoter and ln2 terminator. There is a SwaI restriction site between the corn ubiquitin promoter and the ln2 terminator, which is used to clone the target gene to achieve the purpose of overexpression.

[0043] Step S2 includes the following steps.

[0044] Step S2.1, enzyme digestion of vector plasmid.

[0045] In step S2.1, the enzyme digestion system includes: 10×Fastdigestbuffer 2.5μl; SwaI 0.8 μl; Plasmid 14 μl; Add ddH2O to 30μl; The enzyme digestion was carried out in a water bath at 36.5°C for 1.5 h, and the result was detected by agarose gel electrophoresis. The 15.3 kb band was cut out and recovered.

[0046] Step S2.2, homologous recombination.

[0047] In step S2.2, homologous recombination is performed based on the recombination of the DNA fragment with the homologous sequence at the end of the linearized vector, and the insert fragment is cloned into the linearized vector. The connection system includes: SingleAssemblyCloningMix 4μl; Target fragment 80ng; Linearized vector 40ng; Add ddH2O to 10μl; The reaction system was placed in a 45°C metal bath for 25 min, placed on ice for 2 min, and then transformed into E. coli DH5α.

[0048] Step S2.3, transformation of E. coli DH5α.

[0049] Step S2.3 includes the following steps: Step S2.3.1, place the competent E. coli in a -70°C refrigerator on ice for 3 min, then add the completed ligation system to the competent cell and place it in an ice bath for 25 min; Step S2.3.2, heat shock in 40°C water bath for 45 seconds, ice bath for 1.5 minutes; Step S2.3.3, add 400 μl of LB liquid culture medium into the clean bench, and culture on a shaker at 36.5°C for 45 min; Step S2.3.4: Centrifuge at 4000 rpm for 2 min, discard 320 μl of supernatant, gently pipette and mix the remaining bacterial solution, spread it evenly on LB solid culture medium with Kan resistance, and culture it in a 36.5°C incubator overnight.

[0050] Step S2.4: Colony PCR identification of Escherichia coli.

[0051] Step S2.4 includes the following steps: Step S2.4.1, pick a single colony from the culture medium and streak it on a new culture medium, and take a preset amount of colonies for PCR identification; Step S2.4.2: After the PCR is completed, agarose gel electrophoresis is performed for identification, and positive colonies are selected in LB liquid culture medium and cultured overnight at 36.5°C in a shaking incubator. The bacteria are incubated and plasmids are extracted the next day.

[0052] Step S2.5, plasmid extraction.

[0053] Step S2.5 includes the following steps: Step S2.5.1, collect the positive clones cultured overnight, centrifuge at 12800 rpm for 2 min, and discard the supernatant; Step S2.5.2, add 220 μl of buffer P1 solution and vortex to fully suspend the bacteria; Step S2.5.3, add 220 μl of buffer P2 solution and mix slowly; Step S2.5.4, add 320 μl of buffer N3 solution and mix immediately; Step S2.5.5, centrifuge at 12800 rpm for 8 min, aspirate the supernatant into the adsorption column, let stand for 1.5 min, centrifuge at 12800 rpm for 0.5 min, and discard the waste liquid in the collection tube; Step S2.5.6, add 380 μl of Wash Buffer to the adsorption column, centrifuge at 12800 rpm for 0.5 min, and discard the waste liquid; centrifuge at 12800 rpm for 2 minutes to remove the residual Wash Buffer; Step S2.5.7, transfer the adsorption column into a new 1.5 ml tube, open the lid, let it stand for 4 min, and remove the remaining Wash Buffer; Step S2.5.8, add 40 μl of elution buffer, incubate at room temperature for two minutes, centrifuge at 12800 rpm for 0.5 min; store at -18°C.

[0054] Step S2.6, plasmid sequencing.

[0055] The bacterial liquid or plasmid was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the sequencing results were finally analyzed using SnapGene software.

[0056] Step S2.7, transform Agrobacterium EHA105.

[0057] Step S2.7 includes the following steps: Step S2.7.1, thaw the competent Agrobacterium on ice for 2 min, and add 1.5 μl of the correctly sequenced plasmid DNA; Step S2.7.2, ice bath for 4 min, liquid nitrogen freezing for 4 min, 36.5°C water bath for 4 min, stand on ice for 1.5 min, add 350 μl of YEP liquid culture medium in a sterile clean bench, and shake culture at 26°C for 1.5 hours; Step S2.7.3, centrifuge at 3800 rpm for 1.5 min, discard 300 μl YEP liquid culture medium, evenly spread the remaining bacterial liquid on YEP solid culture medium with Kan resistance, and culture in a 26°C incubator overnight.

[0058] Step S3: wheat genetic transformation.

[0059] Step S3 includes the following steps: The wheat variety Fielder was used as the recipient, and the growth conditions were 15 h of light, 24 °C, 9 h of darkness, 18 °C, and topdressing was applied once at the tillering stage and the grain filling stage; Step S3.1, material disinfection and sterilization: peel the wheat kernels, disinfect them with 75% alcohol for 0.5 min, 10% sodium hypochlorite solution for 8 min, and wash them with sterile water twice; Step S3.2, embryo processing: peel off the embryos under a microscope and place them in the infection solution, wash them twice in the infection solution, add the infection solution again, and centrifuge at 3°C ​​for 8 minutes; Step S3.3, bacterial solution preparation: pick a single colony and shake it overnight at 26°C, centrifuge it and discard the supernatant, then add the invasion dye solution to resuspend it; Step S3.4, infecting young embryos; aspirating the infection solution in the young embryo tube, adding the resuspended bacterial solution, and standing at room temperature for 4 minutes; Step S3.5, co-cultivation: embryos are transferred to co-cultivation medium and cultured in the dark at 21° C. for 1 day; Step S3.6, recovery culture: excise the embryonic axis, transfer to recovery medium, and culture in the dark at 23°C for 4 days; Step S3.7, screening culture: transfer to screening medium 1, 23°C, and culture in the dark for 1.5 weeks, and then transfer to screening medium 2, 23°C, and culture in the dark for 2 weeks; Step S3.8, differentiation culture: the material is transferred to differentiation medium and cultured at 23°C under light for 1.5 weeks; Step S3.9, rooting culture: the material was transferred to the rooting medium and cultured at 23°C under light for 1.5 weeks.

[0060] Example 5, a genetic transformation method for improving wheat transformation efficiency, comprising the following steps: Step S1, obtaining and amplifying a gene fragment of TaLOGL9 or TaARF11, the nucleotide sequence of the TaLOGL9 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the TaARF11 gene is shown in SEQ ID NO.3.

[0061] Specifically, based on the genome information of the wheat variety Chinese Spring, the sequence information of genes TaLOGL9 and TaARF11 was searched on the wheat genome website, primers were designed, and the corresponding gene fragments were amplified using the DNA of the wheat variety Chinese Spring as a template.

[0062] The sequence of primer TaLOGL9F is shown as SEQ ID NO.5.

[0063] The sequence of primer TaLOGL9R is shown in SEQ ID NO.6.

[0064] The sequence of primer TaARF11F is shown in SEQ ID NO.7.

[0065] The sequence of primer TaARF11R is shown in SEQ ID NO.8.

[0066] The TaGRF4-GIF1 gene fragment was amplified from the vector pGRF4-GIF1.

[0067] The sequence of primer TaGRF4F is shown in SEQ ID NO.9.

[0068] The sequence of primer TaGIF1R is shown as SEQ ID NO.10.

[0069] Step S1 includes the following steps.

[0070] Step S1.1, cloning the target gene.

[0071] Step S1.1 includes the following steps.

[0072] Step S1.1.1: Amplify the target fragment using a high-fidelity enzyme. The amplification system includes: PrimeSTARMaxPremix 15μl; Primer F 1 μl; Primer R 1 μl; Target gene 1 μl; Add ddH2O to 30μl; The PCR amplification reaction program was as follows: pre-denaturation at 98°C for 3 min, denaturation at 98°C for 10 s, annealing at 60°C for 10 s, extension at 70°C for 30 s, for a total of 35 cycles, post-extension at 72°C for 10 min, and storage at 16°C; Step S1.1.2: After the PCR amplification is completed, agarose gel electrophoresis is performed, and the band of a preset size is cut out and recovered and purified.

[0073] Step S1.2, recovering the target gene.

[0074] Step S1.2 includes the following steps: Step S1.2.1, use a gel detector to cut the target band and place it in a 1.5 ml centrifuge tube, add 300 μl of Binding Buffer, heat the centrifuge tube in a 55°C metal bath, and shake the centrifuge tube every 5 minutes to completely dissolve the gel; Step S1.2.2, after the solution is cooled to room temperature, transfer the solution to the adsorption column, let it stand for 3 min, centrifuge at 12000 rpm for 1 min at room temperature, discard the waste liquid in the collection tube, add 300 μl of Binding Buffer solution to the adsorption column, let it stand for 3 min, centrifuge at 12000 rpm for 1 min, and discard the waste liquid; Step S1.2.3, add 700ul of Wash Buffer solution to the adsorption column, centrifuge at 13000rpm for 1min, and discard the waste liquid; Step S1.2.4, repeat step S1.2.3; Step S1.2.5, centrifuge at 13000 rpm for 3 min, transfer the adsorption column to another new 1.5 ml centrifuge tube, open the cover of the adsorption column, and let it stand to evaporate the residual Wash Buffer solution; Step S1.2.6, add 30 μl of elution buffer to the adsorption column, let stand for 2 min, centrifuge at 13000 rpm for 1 min, and store at -20°C for later use.

[0075] Step S2, step S2, integrate the gene fragment of TaLOGL9 or TaARF11 into the wheat transformation vector. The p69 plasmid is cut with SwaI, and the plasmid is preserved in this laboratory, mainly including tobacco mosaic virus 35s promoter, glufosinate resistance gene bar, tobacco mosaic virus 35s polyA terminator, corn ubiquitin promoter, GUS gene, nos terminator, corn ubiquitin promoter and ln2 terminator. There is a SwaI restriction site between the corn ubiquitin promoter and the ln2 terminator, which is used to clone the target gene to achieve the purpose of overexpression.

[0076] Step S2 includes the following steps.

[0077] Step S2.1, enzyme digestion of vector plasmid.

[0078] In step S2.1, the enzyme digestion system includes: 10×Fastdigestbuffer 2μl; SwaI 1 μl; Plasmid 15 μl; Add ddH2O to 30μl; The enzyme digestion was carried out at 37°C in a water bath for 2 h, and the fragment was detected by agarose gel electrophoresis. The 15.3 kb band was cut out and recovered.

[0079] Step S2.2, homologous recombination.

[0080] In step S2.2, homologous recombination is performed based on the recombination of the DNA fragment with the homologous sequence at the end of the linearized vector, and the insert fragment is cloned into the linearized vector. The connection system includes: SingleAssemblyCloningMix 5μl; Target fragment 100ng; Linearized vector 50ng; Add ddH2O to 10 μl; The reaction system was placed in a 50°C metal bath for 30 min, placed on ice for 3 min, and then transformed into E. coli DH5α.

[0081] Step S2.3, transformation of E. coli DH5α.

[0082] Step S2.3 includes the following steps: Step S2.3.1, place the competent E. coli in a -80°C refrigerator on ice for 4 minutes, then add the completed ligation system to the competent cell and place it in an ice bath for 30 minutes; Step S2.3.2, heat shock in 42°C water bath for 50 s, ice bath for 2 min; Step S2.3.3, add 400 μl of LB liquid culture medium into the clean bench, and culture on a shaker at 37°C for 50 min; Step S2.3.4: Centrifuge at 4200 rpm for 3 min, discard 340 μl of supernatant, gently pipette and mix the remaining bacterial solution, spread it evenly on LB solid culture medium with Kan resistance, and culture in a 37°C incubator overnight.

[0083] Step S2.4: Colony PCR identification of Escherichia coli.

[0084] Step S2.4 includes the following steps: Step S2.4.1, pick a single colony from the culture medium and streak it on a new culture medium, and take a preset amount of colonies for PCR identification; Step S2.4.2: After the PCR is completed, agarose gel electrophoresis is performed for identification, and positive colonies are selected in LB liquid culture medium and cultured in a shaking incubator at 37°C overnight. The bacteria are incubated and plasmids are extracted the next day.

[0085] Step S2.5, plasmid extraction.

[0086] Step S2.5 includes the following steps: Step S2.5.1, collect the positive clones cultured overnight, centrifuge at 12900 rpm for 3 min, and discard the supernatant; Step S2.5.2, add 250 μl of buffer P1 solution and vortex to fully suspend the bacteria; Step S2.5.3, add 250 μl of buffer P2 solution and mix slowly; Step S2.5.4, add 350 μl of buffer N3 solution and mix immediately; Step S2.5.5, centrifuge at 12900 rpm for 10 min, aspirate the supernatant into the adsorption column, let stand for 2 min, centrifuge at 12900 rpm for 1 min, and discard the waste liquid in the collection tube; Step S2.5.6, add 400 μl of Wash Buffer to the adsorption column, centrifuge at 12900 rpm for 1 min, and discard the waste liquid; centrifuge at 12900 rpm for 3 minutes to remove the residual Wash Buffer; Step S2.5.7, transfer the adsorption column into a new 1.5 ml tube, open the lid, let it stand for 5 min, and remove the remaining Wash Buffer; Step S2.5.8, add 50 μl of elution buffer, incubate at room temperature for two minutes, centrifuge at 13000 rpm for 1 min; store at -20°C.

[0087] Step S2.6, plasmid sequencing.

[0088] The bacterial liquid or plasmid was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the sequencing results were finally analyzed using SnapGene software.

[0089] Step S2.7, transform Agrobacterium EHA105.

[0090] Step S2.7 includes the following steps: Step S2.7.1, thaw the competent Agrobacterium on ice for 3 min, and add 2 μl of the correctly sequenced plasmid DNA; Step S2.7.2, ice bath for 5 min, liquid nitrogen freezing for 5 min, 37°C water bath for 5 min, stand on ice for 2 min, add 400 μl of YEP liquid culture medium in a sterile clean bench, and shake culture at 28°C for 2 h; Step S2.7.3, centrifuge at 4000 rpm for 2 min, discard 340 μl YEP liquid culture medium, evenly spread the remaining bacterial liquid on YEP solid culture medium with Kan resistance, and culture in a 28°C incubator overnight.

[0091] Step S3: wheat genetic transformation.

[0092] Step S3 includes the following steps: The wheat variety Fielder was used as the recipient, and the growth conditions were 16 h of light, 26 °C, 8 h of darkness, 19 °C, and topdressing was applied once at the tillering stage and the grain filling stage; Step S3.1, material disinfection and sterilization: peel the wheat kernels, disinfect them with 75% alcohol for 1 min, 10% sodium hypochlorite solution for 10 min, and wash them with sterile water 3 times; Step S3.2, embryo processing: peel off the embryos under a microscope and place them in the infection solution, wash them twice in the infection solution, add the infection solution again, and centrifuge at 4°C for 10 minutes; Step S3.3, bacterial solution preparation: pick a single colony and shake it overnight at 28°C, centrifuge it and discard the supernatant, then add the invasion dye solution to resuspend it; Step S3.4, infecting young embryos; aspirating the infection solution in the young embryo tube, adding the resuspended bacterial solution, and standing at room temperature for 5 minutes; Step S3.5, co-cultivation: embryos are transferred to co-cultivation medium and cultured in the dark at 23° C. for 2 days; Step S3.6, recovery culture: excise the embryonic axis, transfer to recovery medium, and culture in the dark at 25°C for 5 days; Step S3.7, screening culture: transfer to screening medium 1, 25°C, and culture in the dark for 2 weeks, and then transfer to screening medium 2, 25°C, and culture in the dark for 3 weeks; Step S3.8, differentiation culture: the material is transferred to differentiation medium and cultured at 25°C under light for 2 weeks; Step S3.9, rooting culture: the material was transferred to the rooting medium and cultured at 25° C. under light for 2 weeks.

[0093] Example 6, a genetic transformation method for improving wheat transformation efficiency, comprising the following steps: Step S1, obtaining and amplifying a gene fragment of TaLOGL9 or TaARF11, the nucleotide sequence of the TaLOGL9 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the TaARF11 gene is shown in SEQ ID NO.3.

[0094] Specifically, based on the genome information of the wheat variety Chinese Spring, the sequence information of genes TaLOGL9 and TaARF11 was searched on the wheat genome website, primers were designed, and the corresponding gene fragments were amplified using the DNA of the wheat variety Chinese Spring as a template.

[0095] The sequence of primer TaLOGL9F is shown as SEQ ID NO.5.

[0096] The sequence of primer TaLOGL9R is shown in SEQ ID NO.6.

[0097] The sequence of primer TaARF11F is shown in SEQ ID NO.7.

[0098] The sequence of primer TaARF11R is shown in SEQ ID NO.8.

[0099] The TaGRF4-GIF1 gene fragment was amplified from the vector pGRF4-GIF1.

[0100] The sequence of primer TaGRF4F is shown in SEQ ID NO.9.

[0101] The sequence of primer TaGIF1R is shown as SEQ ID NO.10.

[0102] Step S1 includes the following steps.

[0103] Step S1.1, cloning the target gene.

[0104] Step S1.1 includes the following steps.

[0105] Step S1.1.1: Amplify the target fragment using a high-fidelity enzyme. The amplification system includes: PrimeSTARMaxPremix 16μl; Primer F 1.2 μl; Primer R 1.2 μl; Target gene 1.2 μl; Add ddH2O to 30μl; The PCR amplification reaction program was as follows: pre-denaturation at 100°C for 4 min, denaturation at 100°C for 12 s, annealing at 65°C for 12 s, extension at 75°C for 35 s, for a total of 40 cycles, post-extension at 74°C for 12 min, and storage at 18°C; Step S1.1.2: After the PCR amplification is completed, agarose gel electrophoresis is performed, and the band of a preset size is cut out and recovered and purified.

[0106] Step S1.2, recovering the target gene.

[0107] Step S1.2 includes the following steps: Step S1.2.1, use a gel detector to cut the target band and place it in a 1.5 ml centrifuge tube, add 350 μl of Binding Buffer, heat the centrifuge tube in a 60°C metal bath, and shake the centrifuge tube every 6 minutes to completely dissolve the gel; Step S1.2.2, after the solution is cooled to room temperature, transfer the solution to the adsorption column, let it stand for 4 min, centrifuge at 13000 rpm for 1.5 min at room temperature, discard the waste liquid in the collection tube, add 350 μl of Binding Buffer solution to the adsorption column, let it stand for 4 min, centrifuge at 13000 rpm for 1.5 min, and discard the waste liquid; Step S1.2.3, add 800ul of Wash Buffer solution to the adsorption column, centrifuge at 14000rpm for 1.5min, and discard the waste liquid; Step S1.2.4, repeat step S1.2.3; Step S1.2.5, centrifuge at 14000 rpm for 4 min, transfer the adsorption column to another new 1.5 ml centrifuge tube, open the cover of the adsorption column, and let it stand to evaporate the residual Wash Buffer solution; Step S1.2.6, add 35 μl of elution buffer to the adsorption column, let stand for 3 min, centrifuge at 14000 rpm for 1.5 min, and store at -22°C for later use.

[0108] Step S2, step S2, integrate the gene fragment of TaLOGL9 or TaARF11 into the wheat transformation vector. The p69 plasmid is cut with SwaI, and the plasmid is preserved in this laboratory, mainly including tobacco mosaic virus 35s promoter, glufosinate resistance gene bar, tobacco mosaic virus 35s polyA terminator, corn ubiquitin promoter, GUS gene, nos terminator, corn ubiquitin promoter and ln2 terminator. There is a SwaI restriction site between the corn ubiquitin promoter and the ln2 terminator, which is used to clone the target gene to achieve the purpose of overexpression.

[0109] Step S2 includes the following steps.

[0110] Step S2.1, enzyme digestion of vector plasmid.

[0111] In step S2.1, the enzyme digestion system includes: 10×Fastdigestbuffer 3.5μl; SwaI 1.2 μl; Plasmid 16 μl; Add ddH2O to 30μl; The enzyme digestion was carried out at 37.5℃ in a water bath for 2.5h, and the result was detected by agarose gel electrophoresis. The 15.3kb band was cut out and recovered.

[0112] Step S2.2, homologous recombination.

[0113] In step S2.2, homologous recombination is performed based on the recombination of the DNA fragment with the homologous sequence at the end of the linearized vector, and the insert fragment is cloned into the linearized vector. The connection system includes: SingleAssemblyCloningMix 6μl; Target fragment 120ng; Linearized vector 60ng; Add ddH2O to 10 μl; The reaction system was placed in a 55°C metal bath for 35 min, placed on ice for 4 min, and then transformed into E. coli DH5α.

[0114] Step S2.3, transformation of E. coli DH5α.

[0115] Step S2.3 includes the following steps: Step S2.3.1, place the competent E. coli in a -90°C refrigerator on ice for 5 min, then add the completed ligation system to the competent cell and place it on ice for 35 min; Step S2.3.2, heat shock in 44°C water bath for 55 s, ice bath for 2.5 min; Step S2.3.3, add 400 μl of LB liquid culture medium into the clean bench, and culture on a shaker at 37.5°C for 55 min; Step S2.3.4: Centrifuge at 4400 rpm for 4 min, discard 360 μl of supernatant, gently pipette and mix the remaining bacterial solution, spread it evenly on LB solid culture medium with Kan resistance, and culture in a 37.5°C incubator overnight.

[0116] Step S2.4: Colony PCR identification of Escherichia coli.

[0117] Step S2.4 includes the following steps: Step S2.4.1, pick a single colony from the culture medium and streak it on a new culture medium, and take a preset amount of colonies for PCR identification; Step S2.4.2: After the PCR is completed, agarose gel electrophoresis is performed for identification, and positive colonies are selected in LB liquid culture medium and cultured overnight at 37.5°C in a shaking incubator. The bacteria are incubated and plasmids are extracted the next day.

[0118] Step S2.5, plasmid extraction.

[0119] Step S2.5 includes the following steps: Step S2.5.1, collect the positive clones cultured overnight, centrifuge at 13000 rpm for 4 min, and discard the supernatant; Step S2.5.2, add 280 μl of buffer P1 solution and vortex to fully suspend the bacteria; Step S2.5.3, add 280 μl of buffer P2 solution and mix slowly; Step S2.5.4, add 380 μl of buffer N3 solution and mix immediately; Step S2.5.5, centrifuge at 13000 rpm for 12 min, aspirate the supernatant into the adsorption column, let stand for 2.5 min, centrifuge at 13000 rpm for 1.5 min, and discard the waste liquid in the collection tube; Step S2.5.6, add 420 μl of Wash Buffer to the adsorption column, centrifuge at 13000 rpm for 1.5 min, and discard the waste liquid; centrifuge at 13000 rpm for 4 minutes to remove the residual Wash Buffer; Step S2.5.7, transfer the adsorption column into a new 1.5 ml tube, open the lid, let it stand for 6 min, and remove the remaining Wash Buffer; Step S2.5.8, add 60 μl of elution buffer, incubate at room temperature for two minutes, centrifuge at 13200 rpm for 1.5 min; store at -22°C.

[0120] Step S2.6, plasmid sequencing.

[0121] The bacterial liquid or plasmid was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the sequencing results were finally analyzed using SnapGene software.

[0122] Step S2.7, transform Agrobacterium EHA105.

[0123] Step S2.7 includes the following steps: Step S2.7.1, thaw the competent Agrobacterium on ice for 4 min, and add 2.5 μl of the correctly sequenced plasmid DNA; Step S2.7.2, ice bath for 6 min, liquid nitrogen freezing for 6 min, 37.5°C water bath for 6 min, stand on ice for 2.5 min, add 450 μl of YEP liquid culture medium in a sterile clean bench, and shake culture at 30°C for 2.5 hours; Step S2.7.3, centrifuge at 4200 rpm for 2.5 min, discard 380 μl YEP liquid culture medium, evenly spread the remaining bacterial liquid on YEP solid culture medium with Kan resistance, and culture in a 30°C incubator overnight.

[0124] Step S3: wheat genetic transformation.

[0125] Step S3 includes the following steps: The wheat variety Fielder was used as the recipient, and the growth conditions were 17 h of light, 28 °C, 7 h of darkness, 20 °C, and topdressing was applied once at the tillering stage and the grain filling stage; Step S3.1, material disinfection and sterilization: peel the wheat kernels, disinfect them with 75% alcohol for 1.5 min, 10% sodium hypochlorite solution for 12 min, and wash them with sterile water for 4 times; Step S3.2, embryo processing: peel off the embryos under a microscope and place them in the infection solution, wash them twice in the infection solution, add the infection solution again, and centrifuge at 5°C for 12 minutes; Step S3.3, bacterial solution preparation: pick a single colony and shake it overnight at 30°C, centrifuge it and discard the supernatant, then add the invasion dye solution to resuspend it; Step S3.4, infecting young embryos; aspirating the infection solution in the young embryo tube, adding the resuspended bacterial solution, and standing at room temperature for 6 minutes; Step S3.5, co-cultivation: embryos are transferred to co-cultivation medium and cultured in the dark at 25° C. for 3 days; Step S3.6, recovery culture: excise the embryonic axis, transfer to recovery medium, and culture in the dark at 27°C for 6 days; Step S3.7, screening culture: transfer to screening medium 1, 27°C, and culture in the dark for 2.5 weeks, and then transfer to screening medium 2, 27°C, and culture in the dark for 4 weeks; Step S3.8, differentiation culture: the material is transferred to differentiation medium and cultured at 27° C. under light for 2.5 weeks; Step S3.9, rooting culture: the material was transferred to the rooting medium and cultured at 27° C. under light for 2.5 weeks.

[0126] The following is a schematic table showing the results of wheat transformation efficiency of different genes.

[0127]

[0128] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and substitutions can be made without departing from the technical principles of the present invention. These improvements and substitutions should also be regarded as the scope of protection of the present invention.

Claims

1. Application of TaLOGL9 or TaARF11 gene in improving wheat genetic transformation efficiency, characterized in that: The nucleotide sequence of the TaLOGL9 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the TaARF11 gene is shown in SEQ ID NO.

3.

2. Use of a protein encoded by TaLOGL9 or TaARF11 gene in improving the conversion efficiency of nucleic acid molecules into wheat genome, characterized in that: The amino acid sequence of the protein encoded by the TaLOGL9 gene is shown in SEQ ID NO.2, and the amino acid sequence of the protein encoded by the TaARF11 gene is shown in SEQ ID NO.

4.

3. Use of an expression cassette, a recombinant expression vector or a recombinant bacterium having a TaLOGL9 or TaARF11 gene in improving the conversion efficiency of nucleic acid molecules integrated into a wheat genome, characterized in that: The nucleotide sequence of the TaLOGL9 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the TaARF11 gene is shown in SEQ ID NO.

3.

4. A genetic transformation method for improving wheat transformation efficiency, characterized in that: The following steps are involved: Step S1, obtaining and amplifying a gene fragment of TaLOGL9 or TaARF11, wherein the nucleotide sequence of the TaLOGL9 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the TaARF11 gene is shown as SEQ ID NO.3; Step S2, integrating the gene fragment of TaLOGL9 or TaARF11 into the wheat transformation vector; Step S3: wheat genetic transformation.

5. The genetic transformation method for improving wheat transformation efficiency according to claim 4, characterized in that: Step S1 includes the following steps: Step S1.1, cloning the target gene; Step S1.2, recovering the target gene.

6. The genetic transformation method for improving wheat transformation efficiency according to claim 5, characterized in that: Step S1.1 includes the following steps: Step S1.1.1: Amplify the target fragment using a high-fidelity enzyme. The amplification system includes: PrimeSTARMaxPremix 14-16μl; Primer F 0.8-1.2 μl; Primer R 0.8-1.2 μl; Target gene 0.8-1.2 μl; Add ddH2O to 30μl; The PCR amplification reaction program was as follows: pre-denaturation at 96-100°C for 2-4 min, denaturation at 96-100°C for 8-12 s, annealing at 55-65°C for 8-12 s, extension at 65-75°C for 25-35 s, for a total of 30-40 cycles, post-extension at 70-74°C for 8-12 min, and storage at 14-18°C; Step S1.1.2: After the PCR amplification is completed, agarose gel electrophoresis is performed to cut out the band of a preset size and recover and purify it; Step S1.2 includes the following steps: Step S1.2.1, use a gel detector to cut the target band and place it in a 1.5 ml centrifuge tube, add 250-350 μl of Binding Buffer, heat the centrifuge tube in a 50-60°C metal bath, and shake the centrifuge tube every 4-6 minutes to completely dissolve the gel; Step S1.2.2, after the solution is cooled to room temperature, transfer the solution to an adsorption column, let it stand for 2-4 min, centrifuge at 11000-13000 rpm for 0.5-1.5 min at room temperature, discard the waste liquid in the collection tube, add 250-350 μl of Binding Buffer solution to the adsorption column, let it stand for 2-4 min, centrifuge at 11000-13000 rpm for 0.5-1.5 min, and discard the waste liquid; Step S1.2.3, add 600-800ul of Wash Buffer solution to the adsorption column, centrifuge at 12000-14000rpm for 0.5-1.5min, and discard the waste liquid; Step S1.2.4, repeat step S1.2.3; Step S1.2.5, centrifuge at 12000-14000 rpm for 2-4 min, transfer the adsorption column to another new 1.5 ml centrifuge tube, open the cover of the adsorption column, and let it stand to evaporate the residual Wash Buffer solution; Step S1.2.6, add 25-35 μl of elution buffer to the adsorption column, let stand for 1-3 min, centrifuge at 12000-14000 rpm for 0.5-1.5 min, and store at -18--22°C for later use.

7. The genetic transformation method for improving wheat transformation efficiency according to claim 4, characterized in that: Step S2 includes the following steps: Step S2.1, digesting the vector plasmid; Step S2.2, homologous recombination; Step S2.3, transformation of Escherichia coli DH5α; Step S2.4, Colony PCR Identification of Escherichia coli: Step S2.5, plasmid extraction; Step S2.6, plasmid sequencing; Step S2.7, transform Agrobacterium EHA105.

8. The genetic transformation method for improving wheat transformation efficiency according to claim 7, characterized in that: In step S2.1, the enzyme digestion system includes: 10×Fastdigestbuffer 2.5-3.5μl; SwaI 0.8-1.2 μl; Plasmid 14-16 μl; Add ddH2O to 30μl; Digest in a water bath at 36.5-37.5℃ for 1.5-2.5h, detect by agarose gel electrophoresis, cut out the 15.3kb band and recover; In step S2.2, homologous recombination is performed based on the recombination of the DNA fragment with the homologous sequence at the end of the linearized vector, and the insert fragment is cloned into the linearized vector. The connection system includes: SingleAssemblyCloningMix 4-6μl; Target fragment 80-120ng; Linearized vector 40-60ng; Add ddH2O to 10μl; The reaction system was placed in a 45-55°C metal bath for 25-35 min, placed on ice for 2-4 min, and then transformed into E. coli DH5α.

9. The genetic transformation method for improving wheat transformation efficiency according to claim 7, characterized in that: Step S2.3 includes the following steps: Step S2.3.1, place the competent E. coli in a -70--90°C refrigerator on ice for 3-5 minutes, then add the completed ligation system to the competent cell and place it in an ice bath for 25-35 minutes; Step S2.3.2, heat shock in 40-44°C water bath for 45-55 seconds, ice bath for 1.5-2.5 minutes; Step S2.3.3, add 400 μl of LB liquid culture medium into the clean bench, and culture on a shaker at 36.5-37.5°C for 45-55 min; Step S2.3.4, centrifuge at 4000-4400 rpm for 2-4 min, discard 320-360 μl of the supernatant, pipette and mix the remaining bacterial solution, spread it evenly on LB solid medium with Kan resistance, and culture it in an incubator at 36.5-37.5°C overnight; Step S2.4 includes the following steps: Step S2.4.1, pick a single colony from the culture medium and streak it on a new culture medium, and take a preset amount of colonies for PCR identification; Step S2.4.2: After PCR, perform agarose gel electrophoresis to identify, select positive colonies in LB liquid culture medium, culture them in a shaking incubator at 36.5-37.5°C overnight, and incubate the bacteria and extract the plasmid the next day; Step S2.5 includes the following steps: Step S2.5.1, collect the positive clones cultured overnight, centrifuge at 12800-13000 rpm for 2-4 min, and discard the supernatant; Step S2.5.2, add 220-280 μl of buffer P1 solution and vortex to fully suspend the bacteria; Step S2.5.3, add 220-280 μl of buffer P2 solution and mix slowly; Step S2.5.4, add 320-380 μl of buffer N3 solution and mix immediately; Step S2.5.5, centrifuge at 12800-13000 rpm for 8-12 min, aspirate the supernatant into the adsorption column, let stand for 1.5-2.5 min, centrifuge at 12800-13000 rpm for 0.5-1.5 min, and discard the waste liquid in the collection tube; Step S2.5.6, add 380-420 μl of Wash Buffer to the adsorption column, centrifuge at 12800-13000 rpm for 0.5-1.5 min, and discard the waste liquid; centrifuge at 12800-13000 rpm for 2-4 minutes to remove the residual Wash Buffer; Step S2.5.7, transfer the adsorption column into a new 1.5 ml tube, open the lid, let it stand for 4-6 minutes, and remove the remaining Wash Buffer; Step S2.5.8, add 40-60 μl of elution buffer, incubate at room temperature for two minutes, centrifuge at 12800-13200 rpm for 0.5-1.5 min; store at -18--22°C; Step S2.7 includes the following steps: Step S2.7.1, thaw the competent Agrobacterium on ice for 2-4 minutes, and add 1.5-2.5 μl of the correctly sequenced plasmid DNA; Step S2.7.2, ice bath for 4-6 min, liquid nitrogen freezing for 4-6 min, 36.5-37.5°C water bath for 4-6 min, stand on ice for 1.5-2.5 min, add 350-450 μl of YEP liquid culture medium in a sterile clean bench, and culture at 26-30°C with shaking for 1.5-2.5 hours; Step S2.7.3, centrifuge at 3800-4200 rpm for 1.5-2.5 min, discard 300-380 μl YEP liquid culture medium, evenly spread the remaining bacterial liquid on YEP solid culture medium with Kan resistance, and culture in an incubator at 26-30°C overnight.

10. The genetic transformation method for improving wheat transformation efficiency according to claim 4, characterized in that: Step S3 includes the following steps: The wheat variety Fielder was used as the recipient, and the growth conditions were 15-17 h of light, 24-28 °C, 7-9 h of darkness, 18-20 °C, and topdressing was applied once at the tillering stage and the grain filling stage; Step S3.1, material disinfection and sterilization: peel the wheat kernels, disinfect them with 75% alcohol for 0.5-1.5 min, 10% sodium hypochlorite solution for 8-12 min, and wash them with sterile water 2-4 times; Step S3.2, embryo processing: peel off the embryos under a microscope and place them in the infection solution, wash them twice in the infection solution, add the infection solution again, and centrifuge at 3-5°C for 8-12 minutes; Step S3.3, bacterial solution preparation: pick a single colony and shake it overnight at 26-30°C, centrifuge it and discard the supernatant, then add the invasion dye solution to resuspend it; Step S3.4, infecting young embryos; aspirating the infection solution in the young embryo tube, adding the resuspended bacterial solution, and standing at room temperature for 4-6 minutes; Step S3.5, co-cultivation: embryos are transferred to co-cultivation medium, cultured in the dark at 21-25° C. for 1-3 days; Step S3.6, recovery culture: excise the embryonic axis, transfer to recovery medium, and culture in the dark at 23-27° C. for 4-6 days; Step S3.7, screening culture: transfer to screening medium 1, 23-27°C, dark culture for 1.5-2.5 weeks, then transfer to screening medium 2, 23-27°C, dark culture for 2-4 weeks; Step S3.8, differentiation culture: the material is transferred to differentiation medium and cultured at 23-27°C under light for 1.5-2.5 weeks; Step S3.9, rooting culture: the material is transferred to the rooting medium and cultured at 23-27° C. under light for 1.5-2.5 weeks.

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