Application of TaARF11 Gene in Improving Wheat Genetic Transformation Efficiency

Abstract

The present invention relates to the field of agricultural technologies. Specifically, it relates to the application of the TaARF11 gene in improving the genetic transformation efficiency of wheat, the application of the protein encoded by the TaARF11 gene in improving the transformation efficiency of nucleic acid molecules integrated into the wheat genome, the application of an expression cassette, recombinant expression vector or recombinant bacterium containing the TaARF11 gene in improving the transformation efficiency of nucleic acid molecules integrated into the wheat genome, and a genetic transformation method for improving the transformation efficiency of wheat. The present invention tested the effect of overexpression of the wheat gene TaARF11 on the genetic transformation efficiency. Using the publicly available TaGRF4-GIF1 and a backbone vector without wheat genes as controls, through the binary vector for overexpression in wheat transformation, genes that promote the genetic transformation efficiency of wheat were discovered. The genetic transformation efficiency of the vector containing the TaARF11 gene was improved to varying degrees, providing important gene resources for wheat genetic transformation and new options for wheat biological breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of agriculture. Specifically, it is about the application of the TaARF11 gene in improving the genetic transformation efficiency of wheat. Background Art

[0002] Transgenic technology has become one of the important methods for plant genetic improvement. Transgenic plants have been widely used globally. For example, countries such as the United States, Brazil, and Argentina have planted a large number of transgenic soybeans, corns, cottons, etc. According to data from the global consulting agency AgbioInvestor, in 2023, the global planting area of transgenic crops increased by 1.9% compared to the previous year, reaching 206.3 million hectares (3.09 billion mu), setting a record high. China has also made important progress in the research and application of transgenic plants. For example, the planting area of transgenic insect-resistant cotton has been continuously expanding, and multiple safety certificates have been issued for transgenic soybeans and corns in China. At the same time, gene editing technology is also gradually expanding its application. For example, multiple gene editing safety certificates for soybeans, corns, rice, and wheat have been issued in China.

[0003] Wheat is closely linked to food security supply, social and economic stability, and human nutrition and health due to its high-yield, high-quality, stable-yield, and efficient production characteristics. However, as an allohexaploid plant, its large genome, rich repetitive sequences, and poor regeneration ability pose great challenges to genetic transformation. The Agrobacterium-mediated method 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] By overexpressing key genes that promote regeneration, the regeneration ability and genetic transformation efficiency of crops can be effectively improved, and this new method has gradually been widely recognized. However, the number of currently isolated regeneration-related genes is still limited. Therefore, further isolating and identifying key genes that promote wheat regeneration has important 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 the TaLOGL9 or TaARF11 gene in improving the genetic transformation efficiency of wheat for the above problems. The genetic transformation efficiency of the transformation vector containing the TaLOGL9 or TaARF11 gene is improved to varying degrees, providing important gene resources for wheat genetic transformation.

[0006] To achieve the above purpose, the present invention discloses:

[0007] Use of TaLOGL9 or TaARF11 gene in improving wheat genetic transformation efficiency, characterized in that the nucleotide sequence of TaLOGL9 gene is as shown in SEQ ID NO.1, and the nucleotide sequence of TaARF11 gene is as shown in SEQ ID NO.3.

[0008] Use of the protein encoded by TaLOGL9 or TaARF11 gene in improving the transformation efficiency of nucleic acid molecules integrated into the wheat genome, characterized in that the amino acid sequence of the protein encoded by TaLOGL9 gene is as shown in SEQ ID NO.2, and the amino acid sequence of the protein encoded by TaARF11 gene is as shown in SEQ ID NO.4.

[0009] Use of the expression cassette, recombinant expression vector or recombinant bacterium with TaLOGL9 or TaARF11 gene in improving the transformation efficiency of nucleic acid molecules integrated into the wheat genome, characterized in that the nucleotide sequence of TaLOGL9 gene is as shown in SEQ ID NO.1, and the nucleotide sequence of TaARF11 gene is as shown in SEQ ID NO.3.

[0010] A genetic transformation method for improving wheat transformation efficiency, the method comprising the following steps:

[0011] Step S1, obtaining and amplifying the gene fragment of TaLOGL9 or TaARF11, the nucleotide sequence of TaLOGL9 gene is as shown in SEQ ID NO.1, and the nucleotide sequence of TaARF11 gene is as shown in SEQ ID NO.3;

[0012] Step S2, integrating the gene fragment of TaLOGL9 or TaARF11 into the wheat transformation vector;

[0013] Step S3, wheat genetic transformation.

[0014] Preferably, step S1 includes the following steps:

[0015] Step S1.1, cloning the target gene;

[0016] Step S1.2, recovering the target gene.

[0017] Preferably, step S1.1 includes the following steps:

[0018] Step S1.1.1, amplifying the target fragment using high-fidelity enzyme, and the amplification system includes:

[0019] PrimeSTAR Max Premix 14 - 16 μl;

[0020] Primer F 0.8 - 1.2 μl;

[0021] Primer R: 0.8 - 1.2 μl;

[0022] Target gene: 0.8 - 1.2 μl;

[0023] Add ddH₂O to make up to 30 μl;

[0024] The PCR amplification reaction program is 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, a total of 30 - 40 cycles, post-extension at 70 - 74 °C for 8 - 12 min, and store at 14 - 18 °C;

[0025] Step S1.1.2: After the PCR amplification is completed, perform agarose gel electrophoresis, cut off the band of the preset size, and recover and purify it;

[0026] Step S1.2 includes the following steps:

[0027] 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 BindingBuffer, heat the centrifuge tube in a metal bath at 50 - 60 °C, shake the centrifuge tube every 4 - 6 minutes to completely dissolve the gel;

[0028] Step S1.2.2: After the solution cools to room temperature, transfer the solution into an adsorption column, let it stand for 2 - 4 min, centrifuge at 11000 - 13000 rpm at room temperature for 0.5 - 1.5 min, pour out the waste liquid in the collection tube, add 250 - 350 μl of BindingBuffer solution to the adsorption column again, let it stand for 2 - 4 min, and centrifuge at 11000 - 13000 rpm for 0.5 - 1.5 min, then pour out the waste liquid;

[0029] Step S1.2.3: Add 600 - 800 μl of WashBuffer solution to the adsorption column, centrifuge at 12000 - 14000 rpm for 0.5 - 1.5 min, and pour out the waste liquid;

[0030] Step S1.2.4: Repeat Step S1.2.3;

[0031] 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 lid of the adsorption column, and let it stand to volatilize the residual WashBuffer solution;

[0032] Step S1.2.6: Suspendedly add 25 - 35 μl of elution Buffer onto the adsorption column, let it stand for 1 - 3 min, centrifuge at 12000 - 14000 rpm for 0.5 - 1.5 min, and store it at -18 - -22 °C for standby.

[0033] Preferably, step S2 includes the following steps:

[0034] Step S2.1: Digest the vector plasmid.

[0035] Step S2.2: Homologous recombination.

[0036] Step S2.3: Transform Escherichia coli DH5α.

[0037] Step S2.4: Colony PCR identification of Escherichia coli:

[0038] Step S2.5: Plasmid extraction;

[0039] Step S2.6: Plasmid sequencing;

[0040] Step S2.7: Transform Agrobacterium tumefaciens EHA105.

[0041] Preferably, in step S2.1, the digestion system includes:

[0042] 10×Fastdigest buffer 2.5 - 3.5 μl;

[0043] SwaI 0.8 - 1.2 μl;

[0044] Plasmid 14 - 16 μl;

[0045] Add ddH2O to 30 μl;

[0046] Digest with water bath at 36.5 - 37.5 °C for 1.5 - 2.5 h, detect by agarose gel electrophoresis, cut and recover the 15.3 kb band;

[0047] In step S2.2, for homologous recombination, according to the recombination of homologous sequences at the ends of DNA fragments and the linearized vector, clone the inserted fragment into the linearized vector. The ligation system includes:

[0048] SingleAssembly Cloning Mix 4 - 6 μl;

[0049] Target fragment 80 - 120 ng;

[0050] Linearized vector 40 - 60 ng;

[0051] Add ddH2O to 10 μl;

[0052] Place the reaction system in a metal bath at 45 - 55°C for 25 - 35 min, then place it on ice for 2 - 4 min, and then transform Escherichia coli DH5α.

[0053] Preferably, step S2.3 includes the following steps:

[0054] Step S2.3.1: Place the Escherichia coli competent cells from a -70 to -90°C refrigerator on ice and let them stand for 3 - 5 min. Then add the completed ligation system to the competent cells and incubate on ice for 25 - 35 min.

[0055] Step S2.3.2: Heat shock in a 40 - 44°C water bath for 45 - 55 s, and then incubate on ice for 1.5 - 2.5 min.

[0056] Step S2.3.3: Add 400 μl of LB liquid medium in a laminar flow hood and shake culture at 36.5 - 37.5°C for 45 - 55 min.

[0057] 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, and evenly spread it on an LB solid medium with Kan resistance and incubate overnight in an incubator at 36.5 - 37.5°C.

[0058] Step S2.4 includes the following steps:

[0059] Step S2.4.1: Pick single colonies from the medium and streak them on a new medium, and take a preset amount of colonies for PCR identification.

[0060] Step S2.4.2: After PCR, perform agarose gel electrophoresis identification, select positive colonies into LB liquid medium, shake culture overnight at 36.5 - 37.5°C, and perform bacterial preservation and plasmid extraction the next day.

[0061] Step S2.5 includes the following steps:

[0062] Step S2.5.1: Collect the overnight - cultured positive clone bacterial solution, centrifuge at 12800 - 13000 rpm for 2 - 4 min, and discard the supernatant.

[0063] Step S2.5.2: Add 220 - 280 μl of buffer P1 solution and vortex - shake to fully suspend the bacterial cells.

[0064] Step S2.5.3: Add 220 - 280 μl of buffer P2 solution and mix gently.

[0065] Step S2.5.4: Add 320 - 380 μl of buffer N3 solution and mix immediately.

[0066] Step S2.5.5: Centrifuge at 12,800 - 13,000 rpm for 8 - 12 min, aspirate the supernatant into the adsorption column, let it stand for 1.5 - 2.5 minutes, centrifuge at 12,800 - 13,000 rpm for 0.5 - 1.5 min, and discard the waste liquid in the collection tube;

[0067] Step S2.5.6: Add 380 - 420 μl of Wash Buffer to the adsorption column, centrifuge at 12,800 - 13,000 rpm for 0.5 - 1.5 min, and discard the waste liquid; centrifuge at 12,800 - 13,000 rpm for 2 - 4 minutes to completely remove the residual Wash Buffer;

[0068] Step S2.5.7: Transfer the adsorption column into a new 1.5 ml tube, open the lid, let it stand for 4 - 6 min to remove the remaining Wash Buffer;

[0069] Step S2.5.8: Add 40 - 60 μl of Elution Buffer, incubate at room temperature for two minutes, centrifuge at 12,800 - 13,200 rpm for 0.5 - 1.5 min; store at -18 - -22 °C;

[0070] Step S2.7 includes the following steps:

[0071] Step S2.7.1: Place the Agrobacterium competent cells on ice to thaw for 2 - 4 min, and add 1.5 - 2.5 μl of plasmid DNA with correct sequencing;

[0072] Step S2.7.2: Incubate on ice for 4 - 6 min, freeze in liquid nitrogen for 4 - 6 min, incubate in a water bath at 36.5 - 37.5 °C for 4 - 6 min, let it stand on ice for 1.5 - 2.5 min, and add 350 - 450 μl of YEP liquid medium in a sterile laminar flow hood, then shake and culture at 26 - 30 °C for 1.5 - 2.5 hours;

[0073] Step S2.7.3: Centrifuge at 3,800 - 4,200 rpm for 1.5 - 2.5 min, discard 300 - 380 μl of YEP liquid medium, and evenly spread the remaining bacterial liquid on the YEP solid medium with Kan resistance, then culture overnight in an incubator at 26 - 30 °C.

[0074] Preferably, Step S3 includes the following steps:

[0075] Using the wheat variety Fielder as the receptor, the growth conditions are 15 - 17 h of light, 24 - 28 °C, 7 - 9 h of darkness, 18 - 20 °C, and top - dress once during the tillering stage and the filling stage;

[0076] Step S3.1. Disinfection and sterilization of materials: Shell out wheat grains, disinfect with 75% alcohol for 0.5 - 1.5 min, soak in 10% sodium hypochlorite solution for 8 - 12 min, and wash with sterile water 2 - 4 times.

[0077] Step S3.2. Treatment of young embryos: Under a microscope, shell out young embryos and place them in the infection solution. After washing twice with the infection solution, add the infection solution again and centrifuge at 3 - 5 °C for 8 - 12 min.

[0078] Step S3.3. Preparation of bacterial solution: Pick a single colony and shake the bacteria overnight at 26 - 30 °C. After centrifugation, discard the supernatant and resuspend with the infection solution.

[0079] Step S3.4. Infection of young embryos: Aspirate the infection solution in the young embryo tube, add the resuspended bacterial solution, and let it stand at room temperature for 4 - 6 min.

[0080] Step S3.5. Co - cultivation: Transfer the embryos to the co - cultivation medium and incubate in the dark at 21 - 25 °C for 1 - 3 days.

[0081] Step S3.6. Recovery culture: Excise the hypocotyl, transfer to the recovery medium, and incubate in the dark at 23 - 27 °C for 4 - 6 days.

[0082] Step S3.7. Screening culture: Transfer to screening medium 1 and incubate in the dark at 23 - 27 °C for 1.5 - 2.5 weeks, then transfer to screening medium 2 and incubate in the dark at 23 - 27 °C for 2 - 4 weeks.

[0083] Step S3.8. Differentiation culture: Transfer the materials to the differentiation medium and incubate under light at 23 - 27 °C for 1.5 - 2.5 weeks.

[0084] Step S3.9. Rooting culture: Transfer the materials to the rooting medium and incubate under light at 23 - 27 °C for 1.5 - 2.5 weeks.

[0085] In summary, the beneficial effects of the present invention are as follows: The present invention tested the effects of overexpression of wheat genes TaLOGL9 or TaARF11 on the genetic transformation efficiency. Using the publicly available TaGRF4 - GIF1 and the backbone vector without wheat genes as controls, through the binary vector for overexpression in wheat transformation, genes promoting wheat genetic transformation efficiency were discovered.

[0086] Using young embryos of wheat variety Fielder as explants, by the Agrobacterium - mediated method, the vector containing the target gene was introduced into the wheat genome. The resistant calli 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 vectors containing TaLOGL9 or TaARF11 genes 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

[0087] Figure 1 It is a schematic diagram of the regenerated seedlings of wheat genetic transformation.

[0088] Figure 2 It is a schematic diagram of the wheat transformation vector p69.

[0089] Figure 3 It is a schematic diagram of the wheat transformation vector p52.

[0090] Figure 4 It is a schematic diagram of the wheat transformation vector p58.

[0091] Figure 5 It is a schematic diagram of the wheat transformation vector p59. Specific implementation manners

[0092] The following combines the accompanying drawings and embodiments to further describe the specific implementation manners of the present invention in detail. The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0093] The following further illustrates the present invention in combination with the accompanying drawings and specific implementation manners:

[0094] Example 1, Application of TaLOGL9 or TaARF11 gene in improving wheat genetic transformation efficiency. The nucleotide sequence of TaLOGL9 gene is shown as SEQ ID NO.1, and the nucleotide sequence of TaARF11 gene is shown as SEQ ID NO.3.

[0095] Example 2, Application of the protein encoded by TaLOGL9 or TaARF11 gene in improving the transformation efficiency of nucleic acid molecules integrated into the wheat genome. The amino acid sequence of the protein encoded by TaLOGL9 gene is shown as SEQ ID NO.2, and the amino acid sequence of the protein encoded by TaARF11 gene is SEQ ID NO.4.

[0096] Example 3, Application of the expression cassette, recombinant expression vector or recombinant bacterium with TaLOGL9 or TaARF11 gene in improving the transformation efficiency of nucleic acid molecules integrated into the wheat genome. The nucleotide sequence of TaLOGL9 gene is shown as SEQ ID NO.1, and the nucleotide sequence of TaARF11 gene is shown as SEQ ID NO.3.

[0097] Example 4, A genetic transformation method for improving wheat transformation efficiency, including the following steps:

[0098] Step S1. Obtain and amplify the gene fragments 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.

[0099] Specifically, according to the genomic information of wheat variety Chinese Spring, search for the sequence information of genes TaLOGL9 and TaARF11 on the wheat genome website, design primers, and use the DNA of wheat variety Chinese Spring as a template to amplify the corresponding gene fragments.

[0100] The sequence of primer TaLOGL9F is shown in SEQ ID NO.5.

[0101] The sequence of primer TaLOGL9R is shown in SEQ ID NO.6.

[0102] The sequence of primer TaARF11F is shown in SEQ ID NO.7.

[0103] The sequence of primer TaARF11R is shown in SEQ ID NO.8.

[0104] Amplify the TaGRF4-GIF1 gene fragment from the vector pGRF4-GIF1.

[0105] The sequence of primer TaGRF4F is shown in SEQ ID NO.9.

[0106] The sequence of primer TaGIF1R is shown in SEQ ID NO.10.

[0107] Step S1 includes the following steps.

[0108] Step S1.1. Clone the target gene.

[0109] Step S1.1 includes the following steps.

[0110] Step S1.1.1. Use a high-fidelity enzyme to amplify the target fragment. The amplification system includes:

[0111] PrimeSTAR Max Premix 14 μl;

[0112] Primer F 0.8 μl;

[0113] Primer R 0.8 μl;

[0114] Target gene 0.8 μl;

[0115] Add ddH2O to 30 μl;

[0116] The PCR amplification reaction procedure is 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, a total of 30 cycles, post-extension at 70°C for 8 min, and storage at 14°C;

[0117] Step S1.1.2: After the PCR amplification is completed, perform agarose gel electrophoresis, cut out the band of the preset size, and recover and purify it.

[0118] Step S1.2: Recover the target gene.

[0119] Step S1.2 includes the following steps:

[0120] Step S1.2.1: Use a gel detector to cut out the target band and place it in a 1.5 ml centrifuge tube, add 250 μl of BindingBuffer, heat the centrifuge tube in a metal bath at 50°C, shake the centrifuge tube every 4 minutes to completely dissolve the gel;

[0121] Step S1.2.2: After the solution cools to room temperature, transfer the solution into an adsorption column, let it stand for 2 min, centrifuge at 11000 rpm at room temperature for 0.5 min, pour out the waste liquid in the collection tube, re-add 250 μl of BindingBuffer solution to the adsorption column, let it stand for 2 min, centrifuge at 11000 rpm for 0.5 min, and pour out the waste liquid;

[0122] Step S1.2.3: Add 600 μl of WashBuffer solution to the adsorption column, centrifuge at 12000 rpm for 0.5 min, and pour out the waste liquid;

[0123] Step S1.2.4: Repeat Step S1.2.3;

[0124] 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 lid of the adsorption column, and let it stand to volatilize the residual WashBuffer solution;

[0125] Step S1.2.6: Add 25 μl of elution Buffer to the adsorption column in suspension, let it stand for 1 min, centrifuge at 12000 rpm for 0.5 min, and store at -18°C for standby.

[0126] Step S2: Integrate the gene fragments of TaLOGL9 or TaARF11 into the wheat transformation vector. The p69 plasmid was digested with SwaI. The plasmid was stored in this laboratory and mainly included the tobacco mosaic virus 35s promoter, the glufosinate resistance gene bar, the tobacco mosaic virus 35s polyA terminator, the maize ubiquitin promoter, the GUS gene, the nos terminator, the maize ubiquitin promoter, and the ln2 terminator. There was a SwaI cleavage site between the maize ubiquitin promoter and the ln2 terminator for cloning the target gene to achieve the purpose of overexpression.

[0127] Step S2 includes the following steps.

[0128] Step S2.1: Digest the vector plasmid.

[0129] In step S2.1, the digestion system included:

[0130] 10×Fastdigest buffer 2.5 μl;

[0131] SwaI 0.8 μl;

[0132] Plasmid 14 μl;

[0133] Add ddH2O to 30 μl;

[0134] Digest at 36.5 °C in a water bath for 1.5 h, detect by agarose gel electrophoresis, cut off the 15.3 kb band and recover it.

[0135] Step S2.2: Homologous recombination.

[0136] In step S2.2, homologous recombination was based on the recombination of homologous sequences at the ends of the DNA fragment and the linearized vector to clone the inserted fragment into the linearized vector. The ligation system included:

[0137] SingleAssembly Cloning Mix 4 μl;

[0138] Target fragment 80 ng;

[0139] Linearized vector 40 ng;

[0140] Add ddH2O to 10 μl;

[0141] Place the reaction system in a 45 °C metal bath for 25 min, place on ice for 2 min, and then transform Escherichia coli DH5α.

[0142] Step S2.3: Transformation of Escherichia coli DH5α.

[0143] Step S2.3 includes the following steps:

[0144] Step S2.3.1: Place the Escherichia coli competent cells from a -70°C refrigerator on ice for 3 min, and then add the completed ligation system to the competent cells and incubate on ice for 25 min.

[0145] Step S2.3.2: Heat shock in a 40°C water bath for 45 s and then incubate on ice for 1.5 min.

[0146] Step S2.3.3: Add 400 μl of LB liquid medium in a laminar flow hood and shake culture at 36.5°C for 45 min.

[0147] Step S2.3.4: Centrifuge at 4000 rpm for 2 min, discard 320 μl of the supernatant, gently pipette and mix the remaining bacterial solution, and evenly spread it on an LB solid medium with Kan resistance and incubate overnight in an incubator at 36.5°C.

[0148] Step S2.4: Colony PCR identification of Escherichia coli.

[0149] Step S2.4 includes the following steps:

[0150] Step S2.4.1: Pick a single colony from the medium and streak it on a new medium, and take a preset amount of the colony for PCR identification.

[0151] Step S2.4.2: After PCR, perform agarose gel electrophoresis identification, select positive colonies into LB liquid medium, shake culture overnight at 36.5°C, and perform bacterial preservation and plasmid extraction the next day.

[0152] Step S2.5: Plasmid extraction.

[0153] Step S2.5 includes the following steps:

[0154] Step S2.5.1: Collect the overnight cultured positive clone bacterial solution, centrifuge at 12800 rpm for 2 min, and discard the supernatant.

[0155] Step S2.5.2: Add 220 μl of buffer P1 solution and vortex thoroughly to suspend the bacterial cells.

[0156] Step S2.5.3: Add 220 μl of buffer P2 solution and mix gently.

[0157] Step S2.5.4: Add 320 μl of buffer N3 solution and mix immediately.

[0158] Step S2.5.5: Centrifuge at 12800 rpm for 8 min, pipette the supernatant into the adsorption column, let it stand for 1.5 min, centrifuge at 12800 rpm for 0.5 min, and discard the waste liquid in the collection tube.

[0159] Step S2.5.6: Add 380 μl of Wash Buffer to the adsorption column, centrifuge at 12,800 rpm for 0.5 min, discard the waste liquid; centrifuge at 12,800 rpm for 2 minutes to completely remove the remaining Wash Buffer.

[0160] Step S2.5.7: Transfer the adsorption column to a new 1.5-ml tube, open the lid, let it stand for 4 min to remove the remaining Wash Buffer.

[0161] Step S2.5.8: Add 40 μl of elution Buffer, incubate at room temperature for two minutes, centrifuge at 12,800 rpm for 0.5 min; store at -18°C.

[0162] Step S2.6: Plasmid sequencing.

[0163] Send the bacterial liquid or plasmid to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and finally use SnapGene software for sequencing result analysis.

[0164] Step S2.7: Transform Agrobacterium tumefaciens EHA105.

[0165] Step S2.7 includes the following steps:

[0166] Step S2.7.1: Place the Agrobacterium competent cells on ice to thaw for 2 min, add 1.5 μl of plasmid DNA with correct sequencing.

[0167] Step S2.7.2: Incubate on ice for 4 min, freeze in liquid nitrogen for 4 min, incubate in a 36.5°C water bath for 4 min, let it stand on ice for 1.5 min, add 350 μl of YEP liquid medium in a sterile laminar flow hood, and culture with shaking at 26°C for 1.5 hours.

[0168] Step S2.7.3: Centrifuge at 3,800 rpm for 1.5 min, discard 300 μl of YEP liquid medium, evenly spread the remaining bacterial liquid on the YEP solid medium with Kan resistance, and culture overnight in an incubator at 26°C.

[0169] Step S3: Wheat genetic transformation.

[0170] Step S3 includes the following steps:

[0171] Using wheat variety Fielder as the receptor, with growth conditions of 15 h of light at 24°C, 9 h of darkness at 18°C, and topdressing once during the tillering stage and filling stage.

[0172] Step S3.1: Disinfect and sterilize the materials; peel out the wheat grains, disinfect with 75% alcohol for 0.5 min, 10% sodium hypochlorite solution for 8 min, and wash twice with sterile water.

[0173] Step S3.2, Immature embryo treatment: Under a microscope, dissect the immature embryo and place it in the infection solution. After washing twice with the infection solution, add the infection solution again and centrifuge at 3°C for 8 minutes.

[0174] Step S3.3, Bacterial solution preparation: Pick a single colony and shake it overnight at 26°C. After centrifugation, discard the supernatant and resuspend it with the infection solution.

[0175] Step S3.4, Infecting the immature embryo: Aspirate the infection solution in the embryo tube, add the resuspended bacterial solution, and let it stand at room temperature for 4 minutes.

[0176] Step S3.5, Co-culture: Transfer the embryo to the co-culture medium and incubate it in the dark at 21°C for 1 day.

[0177] Step S3.6, Recovery culture: Excise the hypocotyl and transfer it to the recovery medium, and incubate it in the dark at 23°C for 4 days.

[0178] Step S3.7, Screening culture: Transfer it to the screening medium 1 and incubate it in the dark at 23°C for 1.5 weeks, then transfer it to the screening medium 2 and incubate it in the dark at 23°C for 2 weeks.

[0179] Step S3.8, Differentiation culture: Transfer the material to the differentiation medium and incubate it under light at 23°C for 1.5 weeks.

[0180] Step S3.9, Rooting culture: Transfer the material to the rooting medium and incubate it under light at 23°C for 1.5 weeks.

[0181] Example 5, A genetic transformation method for improving the transformation efficiency of wheat, comprising the following steps:

[0182] Step S1, Obtain and amplify the gene fragments 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.

[0183] Specifically, according to the genomic information of the wheat variety Chinese Spring, search for the sequence information of the genes TaLOGL9 and TaARF11 on the wheat genome website, design primers, and use the DNA of the wheat variety Chinese Spring as a template to amplify the corresponding gene fragments.

[0184] The sequence of primer TaLOGL9F is shown in SEQ ID NO.5.

[0185] The sequence of primer TaLOGL9R is shown in SEQ ID NO.6.

[0186] The sequence of primer TaARF11F is shown in SEQ ID NO.7.

[0187] The sequence of primer TaARF11R is shown in SEQ ID NO.8.

[0188] The TaGRF4-GIF1 gene fragment was amplified from the vector pGRF4-GIF1.

[0189] The sequence of primer TaGRF4F is shown in SEQ ID NO.9.

[0190] The sequence of primer TaGIF1R is shown in SEQ ID NO.10.

[0191] Step S1 includes the following steps.

[0192] Step S1.1: Clone the target gene.

[0193] Step S1.1 includes the following steps.

[0194] Step S1.1.1: Use high-fidelity enzyme to amplify the target fragment. The amplification system includes:

[0195] PrimeSTARMax Premix 15 μl;

[0196] Primer F 1 μl;

[0197] Primer R 1 μl;

[0198] Target gene 1 μl;

[0199] Add ddH2O to 30 μl;

[0200] The PCR amplification reaction program is: 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, a total of 35 cycles, post-extension at 72°C for 10 min, and preservation at 16°C;

[0201] Step S1.1.2: After the PCR amplification is completed, perform agarose gel electrophoresis, cut off the band of the preset size, and recover and purify it.

[0202] Step S1.2: Recover the target gene.

[0203] Step S1.2 includes the following steps:

[0204] Step S1.2.1: Use a gel detector to cut off 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, shake the centrifuge tube every 5 minutes to completely dissolve the gel;

[0205] Step S1.2.2. After the solution is cooled to room temperature, transfer the solution into an adsorption column, let it stand for 3 min, centrifuge at 12,000 rpm at room temperature for 1 min, pour out the waste liquid in the collection tube, add 300 μl of BindingBuffer solution to the adsorption column again, let it stand for 3 min, centrifuge at 12,000 rpm for 1 min, and pour out the waste liquid;

[0206] Step S1.2.3. Add 700 μl of WashBuffer solution to the adsorption column, centrifuge at 13,000 rpm for 1 min, and pour out the waste liquid;

[0207] Step S1.2.4. Repeat Step S1.2.3;

[0208] Step S1.2.5. Centrifuge at 13,000 rpm for 3 min, transfer the adsorption column to another new 1.5 ml centrifuge tube, open the lid of the adsorption column, and let it stand to volatilize the residual WashBuffer solution;

[0209] Step S1.2.6. Add 30 μl of elution Buffer to the adsorption column in suspension, let it stand for 2 min, centrifuge at 13,000 rpm for 1 min, and store at -20 °C for standby.

[0210] Step S2. Integrate the gene fragment of TaLOGL9 or TaARF11 into the wheat transformation vector. Digest the p69 plasmid with SwaI. The plasmid is stored in this laboratory and mainly includes the tobacco mosaic virus 35s promoter, the glufosinate resistance gene bar, the tobacco mosaic virus 35s polyA terminator, the maize ubiquitin promoter, the GUS gene, the nos terminator, the maize ubiquitin promoter and the ln2 terminator. There is a SwaI digestion site between the maize ubiquitin promoter and the ln2 terminator for cloning the target gene to achieve the purpose of overexpression.

[0211] Step S2 includes the following steps.

[0212] Step S2.1. Digest the vector plasmid.

[0213] In Step S2.1, the digestion system includes:

[0214] 10×Fastdigest buffer 2 μl;

[0215] SwaI 1 μl;

[0216] Plasmid 15 μl;

[0217] Add ddH2O to 30 μl;

[0218] Digest with enzymes in a 37°C water bath for 2 h, detect by agarose gel electrophoresis, cut off the 15.3 kb band and recover it.

[0219] Step S2.2, homologous recombination.

[0220] In step S2.2, homologous recombination is based on the recombination of homologous sequences at the ends of DNA fragments and linearized vectors, and the inserted fragment is cloned into the linearized vector. The ligation system includes:

[0221] SingleAssembly Cloning Mix 5 μl;

[0222] Target fragment 100 ng;

[0223] Linearized vector 50 ng;

[0224] Add ddH2O to 10 μl;

[0225] Place the reaction system in a 50°C metal bath for 30 min, place on ice for 3 min, and then transform Escherichia coli DH5α.

[0226] Step S2.3, transformation of Escherichia coli DH5α.

[0227] Step S2.3 includes the following steps:

[0228] Step S2.3.1, Place the Escherichia coli competent cells from the -80°C refrigerator on ice for 4 min, then add the completed ligation system to the competent cells and incubate on ice for 30 min;

[0229] Step S2.3.2, Heat shock in a 42°C water bath for 50 s and incubate on ice for 2 min;

[0230] Step S2.3.3, Add 400 μl of LB liquid medium in a laminar flow hood and shake culture at 37°C for 50 min;

[0231] Step S2.3.4, Centrifuge at 4200 rpm for 3 min, discard 340 μl of the supernatant, gently pipette and mix the remaining bacterial liquid, and spread it evenly on the LB solid medium with Kan resistance and culture overnight in a 37°C incubator.

[0232] Step S2.4, colony PCR identification of Escherichia coli.

[0233] Step S2.4 includes the following steps:

[0234] Step S2.4.1, Pick a single colony from the medium and streak it on a new medium, and take a preset amount of the colony for PCR identification;

[0235] Step S2.4.2: After PCR, perform agarose gel electrophoresis identification. Select positive colonies and culture them overnight in LB liquid medium at 37°C on a shaker. On the next day, preserve the bacteria and extract the plasmid.

[0236] Step S2.5: Plasmid extraction.

[0237] Step S2.5 includes the following steps:

[0238] Step S2.5.1: Collect the overnight cultured positive clone bacterial liquid, centrifuge at 12900 rpm for 3 min, and discard the supernatant.

[0239] Step S2.5.2: Add 250 μl of buffer P1 solution and vortex thoroughly to suspend the bacteria.

[0240] Step S2.5.3: Add 250 μl of buffer P2 solution and mix gently.

[0241] Step S2.5.4: Add 350 μl of buffer N3 solution and mix immediately.

[0242] Step S2.5.5: Centrifuge at 12900 rpm for 10 min, pipette the supernatant into the adsorption column, let it stand for 2 minutes, centrifuge at 12900 rpm for 1 min, and discard the waste liquid in the collection tube.

[0243] 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 remaining Wash Buffer completely.

[0244] Step S2.5.7: Transfer the adsorption column into a new 1.5 ml tube, open the lid, let it stand for 5 min to remove the remaining Wash Buffer.

[0245] 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.

[0246] Step S2.6: Plasmid sequencing.

[0247] Send the bacterial liquid or plasmid to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and finally use SnapGene software to analyze the sequencing results.

[0248] Step S2.7: Transform Agrobacterium tumefaciens EHA105.

[0249] Step S2.7 includes the following steps:

[0250] Step S2.7.1: Place the Agrobacterium competent cells on ice for 3 min to thaw, and add 2 μl of plasmid DNA with correct sequencing.

[0251] Step S2.7.2: Incubate on ice for 5 min, freeze in liquid nitrogen for 5 min, incubate in a water bath at 37 °C for 5 min, let stand on ice for 2 min, and add 400 μl of YEP liquid medium in a sterile laminar flow hood, then culture with shaking at 28 °C for 2 hours.

[0252] Step S2.7.3: Centrifuge at 4000 rpm for 2 min, discard 340 μl of the YEP liquid medium, and evenly spread the remaining bacterial liquid on the YEP solid medium with Kan resistance, then culture overnight in an incubator at 28 °C.

[0253] Step S3: Wheat genetic transformation.

[0254] Step S3 includes the following steps:

[0255] Using the wheat variety Fielder as the receptor, with growth conditions of 16 h of light at 26 °C, 8 h of darkness at 19 °C, and topdressing once during the tillering stage and the filling stage.

[0256] Step S3.1: Disinfect and sterilize the materials; peel out the wheat grains, disinfect with 75% alcohol for 1 min, 10% sodium hypochlorite solution for 10 min, and wash with sterile water 3 times.

[0257] Step S3.2: Treat the immature embryos; under a microscope, peel out the immature embryos and place them in the infection solution. After washing twice in the infection solution, add the infection solution again and centrifuge at 4 °C for 10 min.

[0258] Step S3.3: Prepare the bacterial solution; pick a single colony and culture it with shaking overnight at 28 °C. After centrifugation, discard the supernatant and resuspend with the infection solution.

[0259] Step S3.4: Infect the immature embryos; suck out the infection solution in the embryo tube, add the resuspended bacterial solution, and let stand at room temperature for 5 min.

[0260] Step S3.5: Co-culture; transfer the embryos to the co-culture medium and culture in the dark at 23 °C for 2 days.

[0261] Step S3.6: Recovery culture; excise the hypocotyl, transfer to the recovery medium, and culture in the dark at 25 °C for 5 days.

[0262] Step S3.7: Screening culture; transfer to the screening medium 1 and culture in the dark at 25 °C for 2 weeks, then transfer to the screening medium 2 and culture in the dark at 25 °C for 3 weeks.

[0263] Step S3.8: Differentiation culture; transfer the materials to the differentiation medium and culture with light at 25 °C for 2 weeks.

[0264] Step S3.9: Rooting culture; transfer the materials to the rooting medium and culture them under light at 25°C for 2 weeks.

[0265] Example 6. A genetic transformation method for improving the transformation efficiency of wheat, comprising the following steps:

[0266] Step S1: Obtain and amplify the gene fragments 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.

[0267] Specifically, according to the genomic information of the wheat variety Chinese Spring, search for the sequence information of the genes TaLOGL9 and TaARF11 on the wheat genome website, design primers, and use the DNA of the wheat variety Chinese Spring as a template to amplify the corresponding gene fragments.

[0268] The sequence of primer TaLOGL9F is shown in SEQ ID NO.5.

[0269] The sequence of primer TaLOGL9R is shown in SEQ ID NO.6.

[0270] The sequence of primer TaARF11F is shown in SEQ ID NO.7.

[0271] The sequence of primer TaARF11R is shown in SEQ ID NO.8.

[0272] Amplify the TaGRF4-GIF1 gene fragment from the vector pGRF4-GIF1.

[0273] The sequence of primer TaGRF4F is shown in SEQ ID NO.9.

[0274] The sequence of primer TaGIF1R is shown in SEQ ID NO.10.

[0275] Step S1 includes the following steps.

[0276] Step S1.1: Clone the target gene.

[0277] Step S1.1 includes the following steps.

[0278] Step S1.1.1: Use a high-fidelity enzyme to amplify the target fragment. The amplification system includes:

[0279] PrimeSTARMax Premix 16 μl;

[0280] Primer F 1.2 μl;

[0281] Primer R 1.2 μl;

[0282] 1.2 μl of the target gene;

[0283] Add ddH2O to make up to 30 μl;

[0284] The PCR amplification reaction program is 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, a total of 40 cycles, post-extension at 74°C for 12 min, and storage at 18°C;

[0285] In step S1.1.2, after the PCR amplification is completed, perform agarose gel electrophoresis, cut out the band of the preset size, and recover and purify it.

[0286] Step S1.2: Recover the target gene.

[0287] Step S1.2 includes the following steps:

[0288] 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 BindingBuffer, heat the centrifuge tube in a 60°C metal bath, shake the centrifuge tube every 6 minutes to completely dissolve the gel;

[0289] Step S1.2.2: After the solution cools to room temperature, transfer the solution into the adsorption column, let it stand for 4 min, centrifuge at 13000 rpm at room temperature for 1.5 min, pour out the waste liquid in the collection tube, re-add 350 μl of BindingBuffer solution to the adsorption column, let it stand for 4 min, centrifuge at 13000 rpm for 1.5 min, and pour out the waste liquid;

[0290] Step S1.2.3: Add 800 μl of WashBuffer solution to the adsorption column, centrifuge at 14000 rpm for 1.5 min, and pour out the waste liquid;

[0291] Step S1.2.4: Repeat step S1.2.3;

[0292] 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 lid of the adsorption column, and let it stand to volatilize the residual WashBuffer solution;

[0293] Step S1.2.6: Suspend and add 35 μl of elution Buffer to the adsorption column, let it stand for 3 min, centrifuge at 14000 rpm for 1.5 min, and store at -22°C for standby.

[0294] Step S2: Integrate the gene fragments of TaLOGL9 or TaARF11 into the wheat transformation vector. The p69 plasmid was digested with SwaI. The plasmid was stored in this laboratory and mainly included the tobacco mosaic virus 35s promoter, the glufosinate resistance gene bar, the tobacco mosaic virus 35s polyA terminator, the maize ubiquitin promoter, the GUS gene, the nos terminator, the maize ubiquitin promoter and the ln2 terminator. There was a SwaI digestion site between the maize ubiquitin promoter and the ln2 terminator for cloning the target gene to achieve the purpose of overexpression.

[0295] Step S2 includes the following steps.

[0296] Step S2.1: Digest the vector plasmid.

[0297] In step S2.1, the digestion system includes:

[0298] 10×Fastdigest buffer 3.5 μl;

[0299] SwaI 1.2 μl;

[0300] Plasmid 16 μl;

[0301] Add ddH2O to 30 μl;

[0302] Digest with water bath at 37.5°C for 2.5 h, detect by agarose gel electrophoresis, cut and recover the 15.3 kb band.

[0303] Step S2.2: Homologous recombination.

[0304] In step S2.2, homologous recombination is based on the recombination of homologous sequences at the ends of DNA fragments and linearized vectors to clone the inserted fragment into the linearized vector. The ligation system includes:

[0305] SingleAssembly Cloning Mix 6 μl;

[0306] Target fragment 120 ng;

[0307] Linearized vector 60 ng;

[0308] Add ddH2O to 10 μl;

[0309] Place the reaction system in a 55°C metal bath for 35 min, place on ice for 4 min, and then transform Escherichia coli DH5α.

[0310] Step S2.3: Transformation of Escherichia coli DH5α.

[0311] Step S2.3 includes the following steps:

[0312] Step S2.3.1: Place the Escherichia coli competent cells from a -90°C refrigerator on ice for 5 min, then add the completed ligation system to the competent cells and incubate on ice for 35 min;

[0313] Step S2.3.2: Heat shock in a 44°C water bath for 55 s, then incubate on ice for 2.5 min;

[0314] Step S2.3.3: Add 400 μl of LB liquid medium in a laminar flow hood and shake culture at 37.5°C for 55 min;

[0315] Step S2.3.4: Centrifuge at 4400 rpm for 4 min, discard 360 μl of the supernatant, gently pipette and mix the remaining bacterial solution, and evenly spread it on the LB solid medium with Kan resistance, then culture overnight in an incubator at 37.5°C.

[0316] Step S2.4: Identification of Escherichia coli colonies by colony PCR.

[0317] Step S2.4 includes the following steps:

[0318] Step S2.4.1: Pick a single colony from the medium and streak it on a new medium, and take a preset amount of the colony for PCR identification;

[0319] Step S2.4.2: After PCR, perform agarose gel electrophoresis identification, select positive colonies into LB liquid medium, shake culture overnight at 37.5°C, and perform bacteria preservation and plasmid extraction the next day.

[0320] Step S2.5: Plasmid extraction.

[0321] Step S2.5 includes the following steps:

[0322] Step S2.5.1: Collect the overnight cultured positive clone bacterial solution, centrifuge at 13000 rpm for 4 min, and discard the supernatant;

[0323] Step S2.5.2: Add 280 μl of buffer P1 solution and vortex to fully suspend the bacterial cells;

[0324] Step S2.5.3: Add 280 μl of buffer P2 solution and mix gently;

[0325] Step S2.5.4: Add 380 μl of buffer N3 solution and mix immediately;

[0326] Step S2.5.5: Centrifuge at 13000 rpm for 12 min, pipette the supernatant into the adsorption column, let it stand for 2.5 minutes, centrifuge at 13000 rpm for 1.5 min, and discard the waste liquid in the collection tube;

[0327] Step S2.5.6: Add 420 μl of Wash Buffer into the adsorption column, centrifuge at 13,000 rpm for 1.5 min, and discard the waste liquid; centrifuge at 13,000 rpm for 4 minutes to completely remove the residual Wash Buffer.

[0328] Step S2.5.7: Transfer the adsorption column into a new 1.5-ml tube, open the lid, let it stand for 6 min to remove the remaining Wash Buffer.

[0329] Step S2.5.8: Add 60 μl of elution Buffer, incubate at room temperature for two minutes, centrifuge at 13,200 rpm for 1.5 min; store at -22 °C.

[0330] Step S2.6: Plasmid sequencing.

[0331] Send the bacterial liquid or plasmid to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and finally use SnapGene software to analyze the sequencing results.

[0332] Step S2.7: Transform Agrobacterium tumefaciens EHA105.

[0333] Step S2.7 includes the following steps:

[0334] Step S2.7.1: Place the Agrobacterium competent cells on ice to thaw for 4 min, and add 2.5 μl of plasmid DNA with correct sequencing.

[0335] Step S2.7.2: Incubate on ice for 6 min, freeze in liquid nitrogen for 6 min, water bath at 37.5 °C for 6 min, let it stand on ice for 2.5 min, add 450 μl of YEP liquid medium in a sterile laminar flow hood, and shake culture at 30 °C for 2.5 hours.

[0336] Step S2.7.3: Centrifuge at 4,200 rpm for 2.5 min, discard 380 μl of YEP liquid medium, and evenly spread the remaining bacterial liquid on the YEP solid medium with Kan resistance, and culture overnight in an incubator at 30 °C.

[0337] Step S3: Wheat genetic transformation.

[0338] Step S3 includes the following steps:

[0339] Using wheat variety Fielder as the receptor, the growth conditions are 17 h of light at 28 °C, 7 h of darkness at 20 °C, and topdress once during the tillering stage and the filling stage.

[0340] Step S3.1: Disinfect and sterilize the materials; peel out the wheat grains, disinfect with 75% alcohol for 1.5 min, 10% sodium hypochlorite solution for 12 min, and wash with sterile water 4 times.

[0341] Step S3.2, immature embryo treatment; Under the microscope, the immature embryo is dissected and placed in the infection solution. After washing twice with the infection solution, the infection solution is added again, and centrifuged at 5°C for 12 min.

[0342] Step S3.3, bacterial liquid preparation; Pick a single colony and shake the bacteria overnight at 30°C. After centrifugation, discard the supernatant and resuspend with the infection solution.

[0343] Step S3.4, infecting the immature embryo; Aspirate the infection solution in the immature embryo tube, add the resuspended bacterial liquid, and let it stand at room temperature for 6 min.

[0344] Step S3.5, co-culture; Transfer the embryo to the co-culture medium, and culture it in the dark at 25°C for 3 days.

[0345] Step S3.6, recovery culture; Cut off the hypocotyl, transfer it to the recovery medium, and culture it in the dark at 27°C for 6 days.

[0346] Step S3.7, screening culture; Transfer it to the screening medium 1, culture it in the dark at 27°C for 2.5 weeks, and then transfer it to the screening medium 2, culture it in the dark at 27°C for 4 weeks.

[0347] Step S3.8, differentiation culture; Transfer the material to the differentiation medium, and culture it under light at 27°C for 2.5 weeks.

[0348] Step S3.9, rooting culture; Transfer the material to the rooting medium, and culture it under light at 27°C for 2.5 weeks.

[0349] The following is a schematic table of the wheat transformation efficiency results of different genes.

[0350]

[0351] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and substitutions can be made, and these improvements and substitutions should also be regarded as the protection scope of the present invention.

Claims

1. The application of TaARF11 gene in improving wheat genetic transformation efficiency is characterized by: The nucleotide sequence of the TaARF11 gene is shown in SEQ ID NO.

3.

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

4.

3. The use of the expression cassette, recombinant expression vector or recombinant bacteria of the TaARF11 gene in improving the conversion efficiency of nucleic acid molecules integrated into the wheat genome, characterized in that: 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 TaARF11, wherein the nucleotide sequence of the TaARF11 gene is shown in SEQ ID NO.3; Step S2, integrating the gene fragment of 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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