Method for improving genetic transformation efficiency of wheat by using agrobacterium-mediated method

Abstract

The invention discloses a method for improving wheat genetic transformation efficiency by using an agrobacterium-mediated method, and belongs to the technical field of plant genetic engineering. The TaHRF2 gene and the encoding protein thereof can promote nucleic acid molecules to be introduced into target plants and improve the genetic transformation efficiency of the nucleic acid molecules introduced into the target plants, and the target plants include but are not limited to monocotyledonous plants such as wheat. A TaHRF2 gene high expression vector is introduced into an agrobacterium strain, and an agrobacterium-mediated method is utilized to infect a wheat explant, so that the genetic transformation capability of backbone parents such as Beijing No. 8 and Lu wheat No. 1 is improved, and important information and chassis technical support are provided for establishment of an efficient genetic transformation system of wheat main cultivated varieties difficult to regenerate.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a method for improving wheat genetic transformation efficiency by using an Agrobacterium-mediated method. Background Art

[0002] Agrobacterium is a microorganism commonly found in the soil. It is mainly divided into two categories: Agrobacterium tumefaciens and Agrobacterium rhizogenes. They can infect the injured parts of plants and induce the host plants to produce crown galls or hairy roots. Agrobacterium tumefaciens containing Ti plasmid (tumor-inducing plasmid) can induce host cells to produce crown galls, while Agrobacterium rhizogenes carrying Ri plasmid (root-inducing plasmid) can induce plant cells to produce hairy roots. Both contain T-DNA regions. After Agrobacterium enters cells through infecting plant wounds, it can insert T-DNA into the plant genome and can be stably inherited to offspring through meiosis. With the help of Agrobacterium infection, the transfer and integration of exogenous genes into plant cells is achieved, and then transgenic plants are regenerated through cell and tissue culture techniques. Agrobacterium transformation has a wide range of applications. More than 80% of transgenic plants are mediated by Agrobacterium tumefaciens. It has the advantages of genetic stability, easy transformation of large DNA fragments, and simple operation. At present, genetic transformation systems have been established in many crops, such as rice (Nishimura., 2020), corn (Masters et al., 2020), wheat (Ye et al., 2023), cotton (Zhang., 2019), soybean (Li et al., 2017), etc.

[0003] Common wheat is an allohexaploid and one of the main food sources in many countries in the world, providing more than 20% of energy for humans. Due to the large size of the wheat genome and the complex chromosome inheritance, most genes exist in multiple copies. Therefore, it is difficult to use traditional breeding methods to meet the analysis of wheat gene functions and the improvement of agronomic traits, causing wheat biological breeding research to lag far behind other food crops such as corn and rice. The increasing maturity of transgenic technology and gene editing technology has provided technical support for biological breeding. However, the genetic transformation efficiency of most genotypes of wheat is low, the cycle is long, and it is severely restricted by genotype, which is a major obstacle to the analysis of wheat gene functions and biological breeding. Therefore, improving the efficiency of wheat genetic transformation is crucial for gene function research and crop genetic improvement breeding. Summary of the invention

[0004] In view of the above-mentioned prior art, the object of the present invention is to provide a method for improving the efficiency of wheat genetic transformation by using Agrobacterium-mediated method.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides the use of the TaHRF2 gene in any one of the following (1)-(3):

[0007] (1) Improve the transformation efficiency of nucleic acid molecules into the wheat genome;

[0008] (2) Improve the efficiency of wheat plant regeneration;

[0009] (3) Cultivate transgenic and gene-edited wheat plants;

[0010] The TaHRF2 gene is a DNA molecule as shown in the following i) or ii):

[0011] i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1;

[0012] ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

[0013] Preferably, in the above application, the wheat variety is "Beijing No. 8" or "Lumai No. 1".

[0014] The second aspect of the present invention provides the use of a protein encoded by the TaHRF2 gene in any one of the following (1)-(3):

[0015] (1) Improve the transformation efficiency of nucleic acid molecules into the wheat genome;

[0016] (2) Improve the regeneration efficiency of wheat plants.

[0017] (3) Cultivate transgenic and gene-edited wheat plants.

[0018] Preferably, the protein encoded by the TaHRF2 gene is the protein shown in (A1) or (A2) below:

[0019] (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing;

[0020] (A2) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0021] The third aspect of the present invention provides an expression cassette, a recombinant expression vector or a recombinant bacterium containing the TaHRF2 gene for use in any one of the following (1)-(3):

[0022] (1) Improve the transformation efficiency of nucleic acid molecules into the wheat genome;

[0023] (2) Improve the efficiency of wheat plant regeneration;

[0024] (3) Cultivate transgenic and gene-edited wheat plants.

[0025] A fourth aspect of the present invention provides a method for improving wheat transformation efficiency using Agrobacterium-mediated method, comprising the following steps:

[0026] The TaHRF2 gene is connected to an expression vector to construct a recombinant expression vector, and the recombinant expression vector is transferred into Agrobacterium competent cells to obtain an Agrobacterium strain for transformation;

[0027] Agrobacterium strains are used to infect wheat immature embryos, and transgenic and gene-edited wheat plants are obtained by improving the transformation efficiency.

[0028] Preferably, the expression vector is pUbi110 plasmid.

[0029] Preferably, the Agrobacterium competent cells are Agrobacterium EHA105.

[0030] Beneficial effects of the present invention:

[0031] The present invention discovered for the first time that the TaHRF2 gene can improve the transformation efficiency of the target nucleic acid molecule introduced into the target plant and / or promote the introduction of the nucleic acid molecule into the target plant through the Agrobacterium-mediated method, and the target plant includes but is not limited to monocotyledonous plants such as wheat. The CDS sequence of the TaHRF2 gene is used to construct a high expression vector, which is introduced into the Agrobacterium strain and infects wheat embryos. The results showed that compared with the control vector, the TaHRF2 gene high expression vector can promote the target nucleic acid molecule to enter the target plant. The use of the TaHRF2 gene can improve the genetic transformation efficiency of the target gene integrated into the plant genome, improve the genetic transformation ability of backbone varieties such as Beijing No. 8 and Lumai No. 1, and provide important information and chassis technology support for the establishment of an efficient genetic transformation system for main wheat varieties that are difficult to regenerate, which has important economic value and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the structure of the plant expression vector pUbi110-TaHRF2;

[0033] Figure 2 This is a schematic diagram of the structure of the plant expression vector pUbi110-GUS;

[0034] Figure 3Transgenic resistant plants were obtained by transforming young embryo explants of wheat of different genotypes with plant expression vectors pUbi110-TaHRF2 and pUbi110-GUS. In the figure, A is Beijing No. 8 transformed by pUbi110-GUS vector; B is Beijing No. 8 transformed by pUbi110-TaHRF2 vector; C is Lumai No. 1 transformed by pUbi110-GUS vector; and D is Lumai No. 1 transformed by pUbi110-TaHRF2 vector.

[0035] Figure 4 The candidate transgenic plants were obtained by infecting wheat with the plant expression vector pUbi110-TaHRF2.

[0036] Figure 5 This is the PAT / bar test strip rapid detection result of the candidate transgenic plants of the plant expression vector pUbi110-TaHRF2.

[0037] Figure 6 The results of PCR specific amplification of the bar gene in the candidate transgenic plants obtained by infecting wheat with the plant expression vector pUbi110-TaHRF2. 1-7 in the figure represent the candidate transgenic plants infected with Beijing No. 8, and 8-14 in the figure represent the candidate transgenic plants infected with Lumai No. 1. PC represents the positive plasmid, NC represents the negative control, and M represents the 2000bp molecular weight marker. DETAILED DESCRIPTION

[0038] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0039] As mentioned above, low genetic transformation efficiency and strong genotype dependence are the main obstacles restricting the research and application of wheat gene function. At present, the number of wheat regeneration genes isolated and identified is still very small, and many major wheat varieties and backbone parents cannot be successfully transformed.

[0040] "Beijing No. 8" is a wheat variety bred by Academician Zhuang Qiaosheng, a researcher at the Institute of Crop Sciences of the Chinese Academy of Agricultural Sciences. It has the advantages of early maturity, rust resistance, high yield, and good quality. "Lumai No. 1" is a wheat variety selected and bred by Shandong Agricultural University. It is highly resistant to stripe rust, leaf rust, stem rust, powdery mildew, lodging, dry hot wind, and good yellowing. Although these two wheat varieties have been withdrawn from the market, as wheat varieties that were once widely promoted, dozens of wheat varieties have been bred with them as parents. Using them as basic breeding materials for genetic improvement still has good application prospects.

[0041] However, "Beijing No. 8" and "Lumai No. 1" are wheat varieties that are extremely difficult to transform, and it is difficult to integrate transgenics and edit genes in them.

[0042] Based on this, the present invention conducted an in-depth study on the genetic transformation of "Beijing No. 8" and "Lumai No. 1". The results showed that the genetic transformation efficiency of these two extremely difficult to transform wheat varieties can be significantly improved by transferring the TaHRF2 gene derived from wheat into the wheat varieties "Beijing No. 8" or "Lumai No. 1" using the Agrobacterium-mediated method.

[0043] The nucleotide sequence of the TaHRF2 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the TaHRF2 gene is shown in SEQ ID NO.2; specifically, they are as follows:

[0044] TaHRF2 gene:

[0045] ATGGCGGCGACGGCGACTGCGACGGCGGCGGCGACGAGCGTGGTGACGGGGACGACGCGGTGGTGCCCGACGCCGGAGCAGCTGATGATCCTGGAGGAGATGTACCGCGGCGGGCTGCGCACCCCCAACGCGTCGCAGATCCAGCAGATCACGGCGCACCTGGCCCACTACGGCCGCATCGAGGGCAAGAACGTCTTCTACTGGTTCCAGAACCACAAGGCCCGGGACCGCCAGAAGCTCCGCCGCAGGCTCTGCATGAGCCACCACCTCCTCTCCTGCGCGCACTACTACGCCGCCGCCAACGCCGGCCAGTACCACCAGCAGCAGCAGCTCCTCGGCGCCGGCGCGGTGCCTCCTCCGCTGCTGCAGCACCCGCAGCAGCAGCAGTACTACTCCGCCTCTTGCGCCGGTGGCGGCTACGACCAGCACCTGCTCCCGACGACCGTCCCAGCTTCCGCTTACGCTGCTGCTGCTGGGTACGCCTACCCCTTCGCCGGCGTGCCGGCAAGCCGGTGCGCCGAGCCCTCGCCGCCAAACACCCCGCTCTCCTTCCATCATCAGGGAGGAGGCGTAGTGGGATCGCCGGAGTACTCGCTGGGGAGGCTGGGCAACTTCGGCGTGGTGGAGGACACATGCCGGCCGTCGCGGTACGAGCAGCAGCCGCAGCAGCTGGCCGCGGCGACGGAAGATCAGGCGGCGCCGGTGACGGCGACGGGGCTGTTCTGCCGGCCGCTGAAGACGCTGGACCTCTTCCCCGGCGCGATCAAGGAGGAGCAGCGCGACGTCGCCTAG(SEQ ID NO.1)

[0046] Protein encoded by the TaHRF2 gene:

[0047] MAATATATAAATSVVTGTTRWCPTPEQLMILEEMYRGGLRTPNASQIQQITAHLAHYGRIEGKNVFYWFQNHKARDRQKLRRRLCMSHHLLSCAHYYAAANAGQYHQQQQLLGAGAVPPPLLQHPQQQQYYSA SCAGGGYDQHLLPTTVPASAYAAAAGYAYPFAGVPASRCAEPSPPNTPLSFHHQGGGVVGSPEYSLGRLGNFGVVEDTCRPSRYEQQPQQLAAATEDQAAPVTATGLFCRPLKTLDLFPGAIKEEQRDVA(SEQ ID NO.2)

[0048] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0049] The test materials used in the embodiments of the present invention that are not specifically described are all conventional test materials in the art and can be purchased through commercial channels. The present invention introduces the expression vector into the plant cell, and the introduction methods are all well known to those skilled in the art, including but not limited to: Agrobacterium-mediated method, gene gun bombardment method, pollen tube introduction method, electroporation method and ovary injection method, etc. The selection marker gene used in the present invention is the bar gene, which encodes the phosphinothricin acetyltransferase PAT protein, and other selection marker genes and reporter genes including nptII and hpt can be further used. The screening antibiotic selected by the present invention is phosphinothricin, and the selection of bialaphos and other screening agents can also have the same effect; the specific experimental conditions and methods are not indicated in the embodiments of the present invention, usually according to conventional conditions, such as J. Sambrook et al., ed., Science Press, 2002, Molecular Cloning Experiment Guide (3rd Edition); DL Spector et al., ed., Science Press, 2001, Cell Experiment Guide; or according to the conditions recommended by the manufacturer.

[0050] Example 1: Construction of recombinant expression vector

[0051] 1. Construction of recombinant expression vector pUbi110-TaHRF2:

[0052] Total RNA of wheat line Fielder was extracted using Ultrapure RNA Kit (Chemical Industry, Cat. No.: CW0581M).

[0053] Reference cDNA was reverse transcribed using II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Nanjing Novogene Biotech Co., Ltd., catalog number: R212).

[0054] Using cDNA as template, PCR amplification was performed using primer pairs (upstream primer: 5'-ATGGCGGCGACGGCGACTG-3'; downstream primer: 5'-CTAGGCGACGTCGCGCTGC-3'). The amplification system was 2 μl upstream primer (10 μmol / μl), 2 μl downstream primer (10 μmol / μl), 12.5 μl 2×Phanta Max Master Mix, 1 μl cDNA template, and ddH 2 O to make up the total volume to 25 μl. Amplification conditions: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30 seconds, 59°C annealing for 30 seconds, 72°C extension for 30 seconds, 38 cycles; 72°C extension for 5 minutes.

[0055] The PCR product obtained by amplification was -Blunt3 Cloning Kit (Beijing Quanshijin Biotechnology Co., Ltd., Catalog No.: CB301-01) Operation steps link -Blunt3 vector to obtain pEASY-Blunt3-TaHRF2 vector, and sequenced it.

[0056] The single restriction endonuclease cleavage site (SmaI, SpeI) on the overexpression vector pUbi110 was selected for vector digestion, and the digestion product was recovered by gel according to FastPure Gel DNA Extraction Mini Kit (Nanjing Novogene Biotech Co., Ltd., catalog number: DC301-01).

[0057] Using pEASY-Blunt3-TaHRF2 plasmid as template, homologous primer pairs (upstream primer: 5'-CGACTCTAGAGGATCCCCGGGATGGCGGCGACGGCGACT-3'; downstream primer: 5'-GAATTCCGGCTCGAGACTAGTCTAGGCGACGTCGCGCTG-3') were designed for PCR amplification. The amplification system was 2 μl upstream primer (10 μmol / μl), 2 μl downstream primer (10 μmol / μl), 12.5 μl 2×Phanta Max Master Mix, 1 μl cDNA template, and ddH 2O to make up the total volume to 25 μl. Amplification conditions: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30 seconds, 59°C annealing for 30 seconds, 72°C extension for 30 seconds, 38 cycles; 72°C extension for 5 minutes.

[0058] The amplified PCR product was TM DNA Assembly Mix Plus (Jiangsu Yugong Life Science Co., Ltd.) homologous recombination step was connected with the pUbi110 vector digestion product and sequenced. The correct sequenced single clone was extracted with reference to FastPure Plasmid Mini Kit (Nanjing Novogene Biotech Co., Ltd., Catalog No.: DC201-01) to obtain the recombinant expression vector pUbi110-TaHRF2. The schematic diagram of the vector structure is shown in the figure. Figure 1 shown.

[0059] 2. Construction of control vector pUbi110-GUS:

[0060] Refer to NCBI ( https: / / www.ncbi.nlm.nih.gov / ) website, gene ID: MN266288.1, nucleotides 15108-16919, primer pair (upstream primer: 5'-ATGTTACGTCCTGTAGAA-3'; downstream primer: 5'-TCATTGTTTGCCTCCCTG-3') was used for PCR amplification. The amplification system was 2 μl upstream primer (10 μmol / μl), 2 μl downstream primer (10 μmol / μl), 12.5 μl 2×Phanta Max Master Mix, 1 μl cDNA template, and ddH 2 O to make the total volume 25 μl. Amplification conditions: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 1 minute, 38 cycles; 72°C extension for 5 minutes.

[0061] The PCR product obtained by amplification was -Blunt3 Cloning Kit (Cat. No. CB301-01, Beijing Quanshijin Biotechnology Co., Ltd.) was used to connect the PCR product to obtain pEASY-Blunt3-GUS, which was then sequenced and verified. Sequencing analysis showed that the PCR product was the GUS gene.

[0062] Using pEASY-Blunt3-GUS as template, designed primer pair (upstream primer: 5'-CGACTCTAGAGGATCCCCGGGATGTTACGTCCTGTAGAAACCCCA-3'; downstream primer: 5'-GAATTCCGGCTCGAGACTAGTTTGTTTGCCTCCCTGCTGC-3') for PCR amplification. The amplification system was 2 μl upstream primer (10 μmol / μl), 2 μl downstream primer (10 μmol / μl), 12.5 μl 2×Phanta Max Master Mix, 1 μl cDNA template, and ddH 2 O to make up the total volume to 25 μl. Amplification conditions were: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 1 minute, 38 cycles; 72°C extension for 5 minutes.

[0063] The amplified PCR product was TM DNA Assembly Mix Plus (Jiangsu Yugong Life Science Co., Ltd.) homologous recombination step was connected with the pUbi110 vector digestion product and sequenced. The correct sequenced single clone was extracted with reference to FastPure Plasmid Mini Kit (Nanjing Novogene Biotechnology Co., Ltd., Catalog No.: DC201-01) to obtain the pUbi110-GUS vector. The schematic diagram of the vector structure is shown in the figure. Figure 2 shown.

[0064] Example 2: Agrobacterium-mediated transformation of wheat immature embryos and effect investigation

[0065] 1. Transformation of wheat embryos using Agrobacterium-mediated method:

[0066] The detailed steps and methods of Agrobacterium-mediated transformation of wheat immature embryos refer to Wheat (Triticum aestivum L.) Transformation Using Immature Embryos (Ishida et al., 2015). The basic steps of genetic transformation are as follows:

[0067] The recombinant expression vector pUbi110-TaHRF2 constructed in Example 1 was transformed into Agrobacterium EHA105 competent cells to obtain an Agrobacterium strain for transformation, named pUbi110-TaHRF2 / EHA105. The pUbi110-GUS vector constructed in Example 1 was transformed into Agrobacterium EHA105 competent cells to obtain an Agrobacterium strain for transformation, named pUbi110-GUS / EHA105.

[0068] Three days before infection, pUbi110-TaHRF2 / EHA105 and pUbi110-GUS / EHA105 Agrobacterium were inoculated on YEP solid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, respectively, and cultured in the dark at 28°C incubator for 2 days. A single colony was picked and inoculated into YEP liquid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, and cultured overnight at 28°C 220rpm with shaking; the above Agrobacterium solution was placed in a 2ml centrifuge tube, centrifuged at 6000rpm for 5 minutes, the supernatant was discarded, and the precipitate was resuspended with a resuspension solution to obtain Agrobacterium resuspensions of pUbi110-TaHRF2 / EHA105 and pUbi110-GUS / EHA105, respectively.

[0069] The immature embryos of wheat varieties "Beijing No. 8" and "Lumai No. 1" 14 days after flowering were infected with Agrobacterium resuspensions of pUbi110-TaHRF2 / EHA105 and pUbi110-GUS / EHA105, respectively, and spread with the scutellum side up on WLS-AS medium (1 / 10MS basic medium, 1 / 10MS vitamins, 10 g / L glucose, 100 μM acetosyringone, 8 g / L agarose), and cultured in the dark at 23°C incubator for 2 days.

[0070] After co-culture, the immature embryos were transferred to WLS-Res medium (MS basic medium, MS vitamins, 2,4-D 0.5 mg / L, picloram 2.2 mg / L, glutamine 0.5 g / L, casein 0.1 g / L, MgCl 2 6H 2 O 0.75g / L, maltose 40g / L, AgNO 3 0.85 mg / L, vitamin C 100 mg / L, carbenicillin 250 mg / L, agarose 5 g / L), and cultured in a dark incubator at 25°C for 5 days.

[0071] The callus tissue after recovery culture was transferred to WLS-P5 medium (WLS-Res medium supplemented with PPT 5 mg / L) and cultured in a 25° C. incubator in the dark for 14 days.

[0072] The callus was then transferred to WLS-P10 medium (WLS-Res medium supplemented with 10 mg / L PPT) and cultured in a 25° C. incubator in the dark for 21 days.

[0073] The callus tissue was transferred to LSZ-P5 medium (MS basic medium, LS vitamins, zeatin 5 mg / L, sucrose 20 g / L, carbenicillin 250 mg / L, PPT 5 mg / L, plant gel 3 g / L), and cultured in an incubator at 25°C for 14 days ( Figure 3 ).

[0074] The regenerated buds of wheat callus were transferred to LSF-P5 medium (MS basic medium, LS vitamins, IBA0.2mg / L, sucrose 15g / L, carbenicillin 250mg / L, PPT 5mg / L, plant gel 3g / L), and cultured in a 25°C incubator under light until the root length of the regenerated buds was about 1-2 cm ( Figure 4 ).

[0075] The strong seedlings with roots were transplanted into nutrient soil to obtain resistant seedlings of pUbi110-TaHRF2 and pUbi110-GUS, respectively.

[0076] 2. Rapid detection of candidate transgenic plants by PAT / bar test strips and PCR identification

[0077] 1. Use PAT / bar rapid test paper (Article No.: AA1032-LS, Shanghai Youlong Biotechnology Co., Ltd.) to test resistant plants. The specific steps are as follows:

[0078] Cut an appropriate amount of leaf tissue from the wheat candidate transgenic plants and place it in a 1.5 ml centrifuge tube. Use a grinding pestle to rotate the pestle to crush the leaves, and continue pressing for 20 to 30 seconds. Add 0.20 ml (about 8 drops) of extraction buffer, and repeat the crushing steps to allow the sample and extraction buffer to fully contact and mix. Remove the pestle, take out the rapid test paper from the test paper barrel, and insert it directly into the sample slot (the liquid level to be tested cannot exceed the MAX arrow line), start timing, and the result should be read within 5-8 minutes. The timeout is invalid. The rapid test paper test result is as follows: Figure 5 shown.

[0079] 2. Use the CTAB method (Sambrook and Russell, Molecular Cloning Experiment Guide, 2001) to extract genomic DNA from leaves of wheat plants in the T0 generation that were transformed with pUbi110-TaHRF2 and pUbi110-GUS vectors. Design primers to detect the bar gene, the primer pair sequence (upstream primer: 5'-GGCGGTCTGCACCATCGTCAACCACTAC-3'; downstream primer: 5'-AGTCCAGCTGCCAGAAACCCACGTCATG-3'), the amplified sequence length is 446bp. The amplification system is 1μl upstream primer (10μmol / μl), 1μl downstream primer (10μmol / μl), 10μl 2×Rapid Taq Master Mix, 1μl cDNA template, and ddH 2 O to make up the total volume to 20 μl. Amplification conditions: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 15 seconds, 35 cycles; 72°C extension for 5 minutes.

[0080] PCR identification results Figure 6 As shown, the 446 bp bar gene fragment is absent in non-transgenic and wild-type wheat.

[0081] 3. Statistics of genetic transformation efficiency of different wheat genotypes

[0082] After wheat embryos were infected with Agrobacterium, the induced callus was screened, and the number of resistant callus differentiation formed was counted when transferred to LSF-P5 medium; after PCR identification, the number of positive seedlings was counted, and finally the differentiation rate and transformation efficiency of resistant callus were calculated. The calculation formula is as follows:

[0083] Resistant callus differentiation rate (%) = (number of resistant callus differentiation ÷ total number of young embryos) × 100%;

[0084] Transformation efficiency (%) = (number of positive seedlings ÷ total number of young embryos) × 100%;

[0085] Compared with the control vector pUbi110-GUS, transformation with the pUbi110-TaHRF2 vector can effectively improve the transformation efficiency of wheat. The results are shown in Table 1.

[0086] Table 1: Comparison of transformation efficiency between control vector pUbi110-GUS and pUbi110-TaHRF2 vector

[0087]

[0088] The immature embryos of wheat variety Beijing No. 8 were used as explants for genetic transformation. The differentiation rate and transformation efficiency of resistant callus transformed with pUbi110-TaHRF2 vector were 88.57% and 30.00%, respectively, which were much higher than the 11.97% resistant callus differentiation rate when transformed with control vector pUbi110-GUS, and no transgenic plants were obtained by transforming with control vector. When genetic transformation was carried out with Lumai No. 1 as the recipient, the differentiation rates of resistant callus transformed with pUbi110-TaHRF2 vector and control vector pUbi110-GUS were 66.93% and 3.81%, respectively, and the transformation efficiency of pUbi110-TaHRF2 vector was 14.96%, and no positive transgenic plants were obtained by transforming with control vector pUbi110-GUS. The positive plants obtained by transforming with pUbi110-TaHRF2 vector grew and developed normally.

[0089] From the above results, it can be seen that the TaHRF2 gene can greatly improve the transformation efficiency of wheat and partially solve the genotype dependence problem in wheat genetic transformation.

[0090] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of the TaHRF2 gene in any of the following (1)-(3): (1) Improve the transformation efficiency of nucleic acid molecules into the wheat genome; (2) Improve the efficiency of wheat plant regeneration; (3) Cultivate transgenic and gene-edited wheat plants. The TaHRF2 gene is a DNA molecule as shown in the following i) or ii): i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

2. The use according to claim 1, characterized in that: The wheat variety is "Beijing No. 8" or "Lumai No. 1".

3. Use of the protein encoded by the TaHRF2 gene in any of the following (1)-(3): (1) Improve the transformation efficiency of nucleic acid molecules into the wheat genome; (2) Improve the efficiency of wheat plant regeneration; (3) Cultivate transgenic and gene-edited wheat plants.

4. The use according to claim 3, characterized in that: The protein encoded by the TaHRF2 gene is the protein shown in (A1) or (A2) below: (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing; (A2) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

5. Use of an expression cassette, a recombinant expression vector or a recombinant bacterium containing the TaHRF2 gene in any of the following (1)-(3): (1) Improve the transformation efficiency of nucleic acid molecules into the wheat genome; (2) Improve the efficiency of wheat plant regeneration; (3) Cultivate transgenic and gene-edited wheat plants.

6. A method for improving wheat transformation efficiency using Agrobacterium-mediated method, characterized in that: The following steps are involved: The TaHRF2 gene is connected to an expression vector to construct a recombinant expression vector, and the recombinant expression vector is transferred into Agrobacterium competent cells to obtain an Agrobacterium strain for transformation; Agrobacterium strains are used to infect wheat immature embryos, and transgenic and gene-edited wheat plants are obtained by improving the transformation efficiency.

7. The method according to claim 6, characterized in that The expression vector is pUbi110 plasmid.

8. The method according to claim 6, characterized in that The Agrobacterium competent cells are Agrobacterium EHA105.

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