Gene for improving genetic transformation efficiency of wheat and application thereof

Abstract

The invention discloses a gene for improving genetic transformation efficiency of wheat and application of the gene, and belongs to the technical field of plant genetic engineering. By utilizing the TaHRF1 gene and the encoded protein thereof, exogenous nucleic acid molecules can be promoted to be integrated into a target plant genome, the integration efficiency of the nucleic acid molecules is improved, the growth and development of transformed plants are normal, and the target plants comprise but are not limited to monocotyledonous plants such as wheat. An overexpression vector of the wheat TaHRF1 gene is constructed to infect explants of wheat with different genotypes. Statistical data shows that compared with a control vector, the overexpression vector of the TaHRF1 gene can promote nucleic acid molecules to be integrated into a genome of a target plant. By utilizing the TaHRF1 gene, the genetic transformation efficiency of a target gene introduced into a plant can be improved, the regeneration capacity and transformation efficiency of monocotyledonous plants, especially wheat, are improved, and the plant subjected to genetic transformation grows and develops normally. The invention provides a new important gene resource for overcoming genotype dependence of wheat genetic transformation.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a gene for improving wheat genetic transformation efficiency and an application thereof. Background Art

[0002] Wheat is an important food crop in the world. The high yield, high quality, stable production and high efficiency of wheat are closely related to food security, social and economic stability, and human nutrition and health. Wheat breeding is the main technical means to achieve high yield, high quality, stable production and high efficiency of wheat. Genetic transformation technology and gene editing are important technical means to improve agronomic traits such as crop yield, disease resistance and quality, and have achieved great success in crops such as soybeans, corn, cotton and rapeseed. In contrast, wheat is an allohexaploid plant with a large genome, many repetitive sequences, poor regeneration ability, and difficult genetic transformation, which has become the main reason for the slow progress of wheat biological breeding.

[0003] People have tried to use a variety of transformation methods such as gene gun method, Agrobacterium-mediated method, pollen tube channel method, ultrasonic method, ion beam implantation method, laser microbeam puncture method and PEG (Polyethylene glycol) method to introduce foreign genes into target plants. Agrobacterium-mediated method is currently the most commonly used method for wheat transformation. The type of Agrobacterium is mainly Agrobacterium tumefaciens. This method has the advantages of low cost, simple operation, high efficiency of foreign gene introduction, low copy number and good genetic stability. Gene gun method is a physical transformation method. It introduces foreign genes into organisms through the physical means of high-speed particle bombardment. It has the advantages of being independent of genotype and direct transformation. It is also one of the commonly used transformation methods.

[0004] The type of explants used for plant genetic transformation is also an important factor affecting transformation efficiency. Currently, the commonly used explants mainly include young embryos, mature embryos, anther callus and young panicles. Generally speaking, explants taken from young and early stage plant parts have stronger regeneration and transformation capabilities.

[0005] In recent years, wheat genetic transformation technology has made great breakthroughs. Overexpression of key genes that promote regeneration can improve crop regeneration ability and genetic transformation efficiency. A new method to improve wheat genetic transformation efficiency has gradually been recognized by everyone. However, the number of regeneration-related genes that have been isolated is still small. Therefore, further isolation and identification of key genes that promote wheat regeneration is of great significance for improving crop agronomic traits and promoting biotechnology breeding. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide a gene for improving the genetic transformation efficiency of wheat and its application. The present invention has found that the overexpression of the wheat TaHRF1 gene significantly improves the transformation efficiency of multiple wheat varieties and effectively overcomes the genotype dependence of wheat. The application of TaHRF1 can improve the efficiency of plant genetic transformation, and TaHRF1 can be used as a transformation auxiliary gene to improve the genetic transformation efficiency of crops such as wheat, reduce the cost of genetic transformation, and lay a solid foundation for biotechnology breeding. Technical support.

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

[0008] The first aspect of the present invention provides the use of the TaHRF1 gene in the following (1) or (2):

[0009] (1) Improve the transformation efficiency of nucleic acid molecules into the wheat genome;

[0010] (2) Stable expression of nucleic acid molecules improves wheat plant regeneration efficiency;

[0011] The TaHRF1 gene is a nucleic acid molecule as shown in the following i) or ii) or iii):

[0012] i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1;

[0013] ii) Nucleic acid molecules that have 80% or more homology with the nucleotide sequence of i) and express proteins with the same or similar functions, as well as corresponding alleles, homologous genes, mutant genes and derivative genes;

[0014] iii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

[0015] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0016] The term "homology" as used herein refers to sequence similarity to a natural nucleic acid sequence. Homology can be evaluated using computer software, such as the BLAST algorithm (Altschul et al., 1990. Journal of Molecular Biology 215: 403-410; Karlin and Altschul., 1993. Proceedings of the National Academy of Sciences 90: 5873-5877).

[0017] In the above nucleic acid molecules, the 80% or more homology may be at least 80%, 85%, 90%, 95%, 96%, 98% or 99% homology.

[0018] The second aspect of the present invention provides the use of a protein encoded by the TaHRF1 gene in the following (1) or (2):

[0019] (1) Improve the transformation efficiency of nucleic acid molecules into the wheat genome;

[0020] (2) Stable expression of nucleic acid molecules improves wheat plant regeneration efficiency;

[0021] Preferably, the protein encoded by the TaHRF1 gene is a protein as shown in any one of the following (A1) or (A2) or (A3):

[0022] (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing;

[0023] (A2) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0024] (A3) The encoded protein is similar to the protein shown in SEQ ID NO. 2, or a protein obtained by substitution, deletion or insertion of one, several or dozens of amino acids.

[0025] The proteins described in (A1), (A2) and (A3) can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0026] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. In order to facilitate the purification of the protein in (A1), a tag can be connected to the amino terminus or carboxyl terminus of the protein in (A1). The tag can be Poly-Arg (usually 6 RRRRR), Poly-His (usually 6 HHHHHH), FLAG (DYKDDDDK), Strep-tagII (WSHPQFEK) or c-Myc (EQKLISEEDL).

[0027] The third aspect of the present invention provides the use of an expression cassette, a recombinant expression vector or a recombinant bacterium containing the TaHRF1 gene in the following (1) or (2):

[0028] (1) Improve the transformation efficiency of nucleic acid molecules into the wheat genome;

[0029] (2) Stable expression of nucleic acid molecules improves wheat plant regeneration efficiency;

[0030] The recombinant expression vector can be constructed using existing plant expression vectors. Preferably, the recombinant expression vector containing the TaHRF1 gene is constructed using the pc186 expression vector.

[0031] A fourth aspect of the present invention provides a method for improving the transformation efficiency of a nucleic acid molecule introduced into a target plant, comprising the following steps:

[0032] The TaHRF1 gene and the target nucleic acid molecule are transferred into the target plant to achieve the purpose of improving the transformation efficiency of the target nucleic acid molecule introduced into the target plant;

[0033] The TaHRF1 gene is a nucleic acid molecule as shown in the following i) or ii) or iii):

[0034] i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1;

[0035] ii) Nucleic acid molecules that have 80% or more homology with the nucleotide sequence of i) and express proteins with the same or similar functions, as well as corresponding alleles, homologous genes, mutant genes and derivative genes;

[0036] iii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

[0037] In the above method, the TaHRF1 gene and the target nucleic acid molecule can be transferred into the target plant via one vector or via different vectors.

[0038] Preferably, the TaHRF1 gene and the target nucleic acid molecule are transferred into the target plant via the pc186 expression vector.

[0039] In the above method, the target plants include but are not limited to monocotyledonous plants such as wheat, corn, rice, and barley, and can also be applied to dicotyledonous plants such as soybean and rapeseed.

[0040] Beneficial effects of the present invention:

[0041] The present invention has discovered for the first time that the TaHRF1 gene can improve the efficiency of integrating a target nucleic acid molecule into a target plant and / or promote the integration of nucleic acid molecules into a target plant, and the target plant includes but is not limited to monocotyledonous plants such as wheat. An overexpression vector is constructed using the CDS sequence of the TaHRF1 gene, which is introduced into an Agrobacterium strain and infects young embryos of wheat of different genotypes. The results show that compared with the control vector, the TaHRF1 gene overexpression vector can promote the entry and integration of the target nucleic acid molecule into the genome of the target plant. The use of the TaHRF1 gene can improve the efficiency of integrating the target gene into the plant genome, improve the genetic transformation efficiency of monocotyledonous plants, especially wheat, and the genetically transformed plants grow and develop normally. The present invention has important economic value and social benefits for accelerating the study of plant gene functions and improving crop agronomic traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a schematic diagram of the plant expression vector pc186-TaHRF1 vector structure;

[0043] Figure 2 This is a schematic diagram of the plant expression vector pc186-GUS vector structure;

[0044] Figure 3 The tissue culture process of obtaining resistant seedlings by infecting the immature embryos of wheat variety Liangxing 66 with plant expression vector pc186-TaHRF1 and control vector pc186-GUS. In the figure, A represents callus transformed with pc186-TaHRF1 vector, B represents callus transformed with pc186-TaHRF1 vector and growing resistant seedlings, C represents callus transformed with control vector pc186-GUS vector, and D represents callus transformed with control vector pc186-GUS and growing resistant seedlings.

[0045] Figure 4 The tissue culture process of obtaining resistant seedlings by infecting immature embryos of wheat variety Aifeng 3 with plant expression vector pc186-TaHRF1 and control vector pc186-GUS. In the figure, A represents callus transformed with pc186-TaHRF1 vector, B represents callus transformed with pc186-TaHRF1 vector and growing resistant seedlings, C represents callus transformed with control vector pc186-GUS vector, and D represents callus transformed with control vector pc186-GUS and growing resistant seedlings.

[0046] Figure 5Schematic diagram of the results of PCR-specific amplification of the bar gene in candidate transgenic plants obtained by transforming the plant expression vector pc186-TaHRF1. In the figure, 1-8 represent candidate transgenic plants transformed with Liangxing 66, 9-14 represent candidate transgenic plants transformed with Aifeng 3, PC represents positive plasmid, NC represents negative control, WT represents wild-type control, and M represents 2000bp molecular weight marker.

[0047] Figure 6 The positive plants obtained by transforming Fielder with the plant expression vector pc186-TaHRF1 developed normally. WT represents the wild type control, GUS represents the positive plants of the control vector pc186-GUS, and OE-1, OE-2 and OE-3 represent positive plants. DETAILED DESCRIPTION

[0048] 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.

[0049] As mentioned above, due to the large and complex wheat genome, the large number of repetitive DNA sequences, and the low regeneration ability, the application of genetic transformation and gene editing technology in wheat lags far behind other major crops. Studies have found that the use of regeneration genes can promote the efficiency of plant regeneration and genetic transformation in wheat in vitro tissue culture. However, the number of wheat regeneration genes isolated and identified is still very small; the transformation efficiency of different wheat genotypes varies greatly, and most wheat genotypes cannot be successfully transformed; moreover, the introduction of exogenous wheat regeneration genes may have adverse effects on the agronomic traits of wheat. Therefore, it is very difficult to discover wheat regeneration genes with practical production application value.

[0050] "Liangxing 66" is a wheat variety bred by Shandong Liangxing Seed Co., Ltd. using the varieties Ji 91102 / Ji 935031. On December 13, 2010, it was approved by the Fourth Meeting of the Second National Crop Variety Approval Committee, and the approval number is Guoshenmai 2010004; this variety has the characteristics of high yield, good quality, high resistance to lodging, high yield, and wide suitable area. "Aifeng No. 3" is a wheat variety selected and bred by Academician Zhao Hongzhang of Northwest Agriculture and Forestry University. It is the first semi-dwarf variety promoted on a large scale in the history of wheat production in my country. Both of these wheat varieties have been on the market for many years and are now facing the problem of further genetic improvement of the varieties.

[0051] However, "Liangxing 66" and "Aifeng 3" are wheat varieties that are extremely difficult to transform, and it is difficult to integrate transgenics and edit genes in them.

[0052] Based on this, the present invention conducted an in-depth study on the genetic transformation of "Liangxing 66" and "Aifeng 3". The results showed that the genetic transformation efficiency of these two extremely difficult to transform wheat varieties can be significantly improved by transferring the TaHRF1 gene derived from wheat into the wheat varieties "Liangxing 66" and "Aifeng 3" using the Agrobacterium-mediated method.

[0053] 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.

[0054] 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 method is well known to those skilled in the art, including but not limited to: Agrobacterium-mediated method, gene gun bombardment method, electroporation method, ovary injection method, etc. The 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.

[0055] Example 1: Cloning of TaHRF1 gene and construction of expression vector

[0056] Callus tissues formed by wheat Fielder immature embryo explants induced by tissue culture were collected, and total RNA was first extracted using Ultrapure RNA Kit (Kangwei Century, Catalog No.: CW0581M), and then the extracted RNA was reverse transcribed into cDNA with reference to the FastKing RT Kit (With gDNase) kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., Catalog No.: KR116).

[0057] cDNA was amplified with primers (upstream primer: 5'-ATGCCGCAGACGCCATCGAC-3', SEQ ID NO.3; downstream primer: 5'-CTAGTTTGTGGAGGTGGAGCAA-3', SEQ ID NO.4). The total volume of the amplification system was 25 μl, including 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 7.5 μl ddH2O. The amplification conditions were: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 30 seconds, and 32 cycles; extension at 72°C for 5 minutes; and finally holding at 16°C.

[0058] Reference -Blunt3 Cloning Kit (Beijing Quanshijin Biotechnology Co., Ltd., Catalog No.: CB301-01) Operation steps: Connect the PCR amplification product to -Blunt3 vector to obtain pEASY-Blunt3-TaHRF1 vector, and the results were analyzed after Sanger sequencing.

[0059] After sequencing analysis, the nucleotide sequence of the PCR amplification product is Sequence 1 in the sequence list, and the gene shown by the PCR product is named TaHRF1 gene; the protein encoded by the gene is named TaHRF1, and the amino acid sequence of the protein is Sequence 2 in the sequence list. The details are as follows:

[0060] Sequence 1 (SEQ ID NO.1):

[0061] ATGCCGCAGACGCCATCGACCCGTTGGTGCCCGACGCCTGAGCAGCTGATGATCCT

[0062] GGAGGAGATGTACCGGAGCGGCGTGCGCACACCTAACGCGGCGGAGATCCAGCAG

[0063] ATCACGGCGCACCTCGCCTACTACGGCCGCATCGAGGGAAAGAACGTCTTCTACTG

[0064] GTTCCAGAACCACAAGGCCCGCGAGCGCCAGCGTCTCCGTCGCCGCCTCTGCGCCC

[0065] GCCACCAGCAACCCTCCTCCCCGGCGGCTCCTCCTCCTCCTCCTCCTCCTCATACTG

[0066] GTGCTGCCGGTGGCGGAGGCAATGCTGCTGGTGCTGGTGCGGGCGTGAACGTGATG

[0067] CACCCCGCGGTGATGCAGCTGCACCATCACCACCACACATACGCTACCAGCTGCTT

[0068] CATGGCGCCTCAGGGCTACTTGGAGCAGGAAACAGCAGCAGCAGGAGCTCTTCCAG

[0069] TTTCGGGGTTGGAGTTTGCAGGCAAGACAAGCCAGCAGCAGGAATGGATGGCGCA

[0070] GGAGCAGATGGTGATGGAGAACAGCAACATTAACAACAGTGTAGCAGCAGCTGGA

[0071] GGCAGCTCCGCATCGGCCGGCGGTGGTATGAATAATATGACCCCGCCGCCATGGCC

[0072] ATGCTGCCGGCCGCTCAGAACCCTAGAGCTCTTCCCTACAAAGAGCACCGGTGGCG

[0073] GCCTCAGGGACGAGTGCAGCAGCTCCAAGTCCTCCTCTTGCTCCACCTCCACAAACT

[0074] AG

[0075] Sequence 2 (SEQ ID NO.2):

[0076] MPQTPSTRWCPTPEQLMILEEMYRSGVRTPNAAEIQQITAHLAYYGRIEGKNVFYWFQ

[0077] NHKARERQRLRRRLCARHQQPSSPAAPPPPPPPHTGAAGGGGNAAGAGAGVNVMHPA

[0078] VMQLHHHHHTYATSCFMAPQGYLEQETAAAGALPVSGLEFAGKTSQQQEWMAQEQM

[0079] VMENSNINNSVAAAGGSSASAGGGMNNMTPPPWPCCRPLRTLELFPTKSTGGGLRDEC

[0080] SSSKSSSCSTSTN

[0081] Sequence 1 can also be artificially synthesized and connected to the pEASY-Blunt3 vector to obtain the pEASY-Blunt3-TaHRF1 vector.

[0082] Using pEASY-Blunt3-TaHRF1 as template, designed primer pairs (upstream primer: 5'-CACCATGCCGCAGACGCCATC-3', SEQ ID NO.5; downstream primer: 5'-CTAGTTTGTGGAGGTGGAGCAA-3', SEQ ID NO.4) for PCR amplification. The amplification system was 2μl upstream primer (10μmol / μl), 2μl downstream primer (10μmol / μl), 12.5μl 2×Phanta Max Master Mix, 1μl cDNA template, and ddH2O was added to make the total volume 25μl. The amplification conditions were: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 58℃ annealing for 15 seconds, 72℃ extension for 30 seconds, 32 cycles; 72℃ extension for 5 minutes.

[0083] Refer to pENTR TM Directional Cloning Kit (Thermo Scientific TM , Catalog number: K2400-20SP) operation steps, the amplified PCR product was cloned into the Gateway system, and then sequenced. The single clone with correct sequencing was connected to the pc186 expression vector through LR reaction to obtain the pc186-TaHRF1 vector. The vector structure diagram is shown in Figure 1 shown.

[0084] The pc186-TaHRF1 was transformed into Agrobacterium EHA105 competent cells, and an Agrobacterium strain that can be used for transformation was obtained and named pc186-TaHRF1 / EHA105.

[0085] Example 2: Construction of control vector pc186-GUS

[0086] NCBI ( https: / / www.ncbi.nlm.nih.gov / ) website Sequence ID: MN266288.1, nucleotides 15108-16919, were used as templates, and primer pairs (upstream primer: 5'-ATGTTACGTCCTGTAGAA-3', SEQ ID NO.6; downstream primer: 5'-TCATTGTTTGCCTCCCTG-3', SEQ ID NO.7) were used for PCR amplification. The amplification system included 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 (100-200 ng / μl), and 7.5 μl ddH2O. The amplification conditions were: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, extension at 72°C for 50 seconds, and 32 cycles of denaturation, annealing, and extension; extension at 72°C for 5 minutes; and holding at 16°C.

[0087] Reference -Blunt3 Cloning Kit (Cat. No.: CB301-01, Beijing Quanshijin Biotechnology Co., Ltd.) operation steps: connect the PCR amplification product to the cloning vector Blunt3 to obtain the pEASY-B3-GUS vector, and perform sequencing.

[0088] Sequencing analysis showed that the gene expressed by the PCR product was the GUS gene.

[0089] The GUS gene can also be artificially synthesized and connected to the pEASY-Blunt3 vector to obtain pEASY-Blunt3-GUS.

[0090] Using pEASY-Blunt3-GUS as template, designed primer pair (upstream primer: 5'-CACCATGTTACGTCCTGTAGAA-3', SEQ ID NO.8; downstream primer: 5'-TCATTGTTTGCCTCCCTG-3', SEQID NO.7) for PCR amplification. The amplification system was 2μl upstream primer (10μmol / μl), 2μl downstream primer (10μmol / μl), 12.5μl 2×Phanta Max Master Mix, 1μl cDNA template, and ddH2O was added to make the total volume 25μl. Amplification conditions were: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 58℃ annealing for 15 seconds, 72℃ extension for 50 seconds, 32 cycles; 72℃ extension for 5 minutes.

[0091] The amplified PCR product was referred to pENTR TM Directional Cloning Kit (Cat. No.: K2400-20SP, Thermo Scientific TM ) Operation steps: ligation and sequencing. The correctly sequenced single clone was ligated to the pc186 expression vector through LR reaction to obtain the pc186-GUS vector. The vector structure diagram is shown in Figure 2 shown.

[0092] The pc186-GUS was transformed into Agrobacterium EHA105 competent cells, and an Agrobacterium strain that can be used for transformation was obtained and named pc186-GUS / EHA105.

[0093] Example 3: Agrobacterium-mediated transformation of wheat immature embryos and identification of transgenic plants

[0094] 1. The detailed steps and methods of Agrobacterium-mediated method for wheat immature embryos refer to the method of Ishida et al. (Ishida et al., 2015), and the components of each culture medium are shown in Table 1.

[0095] 1. Three days before infection, inoculate pc186-TaHRF1 / EHA105 and pc186-GUS / EHA105 Agrobacterium on YEP solid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, respectively, and culture in a dark incubator at 28°C for 2 days. Pick a single colony and inoculate it in YEP liquid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, and culture it overnight at 28°C and 220 rpm; put the above Agrobacterium liquid into a sterile 2 ml centrifuge tube, centrifuge at 6000 rpm for 5 minutes, discard the supernatant, resuspend the precipitate with resuspension solution, and obtain Agrobacterium resuspension of pc186-TaHRF1 / EHA105 and pc186-GUS / EHA105, respectively.

[0096] 2. Collect immature embryos (scutellum length is about 2 mm) of wheat of different genotypes about 2 weeks after flowering, infect them with Agrobacterium resuspensions of pc186-TaHRF1 / EHA105 and pc186-GUS / EHA105, respectively, spread them on WLS-AS medium with the scutellum side facing up, and culture them in a dark incubator at 23°C for 2 days.

[0097] 3. Transfer the co-cultured embryos to WLS-Res medium and culture in a dark incubator at 25°C for 5 days.

[0098] 4. Transfer the callus tissue after recovery culture to WLS-P5 medium and culture it in a dark incubator at 25°C for 2 weeks.

[0099] 5. Transfer the callus to WLS-P10 medium and culture in a dark incubator at 25°C for 3 weeks.

[0100] 6. Transfer the above callus to LSZ-P5 medium and culture in a 25°C incubator for 2 weeks. Figure 3 and Figure 4 ).

[0101] 7. Transfer the regenerated resistant buds of wheat to LSF-P5 medium and culture them in a 25°C incubator under light until the roots of the regenerated buds are about 1-2 cm long.

[0102] 8. Transplant the strong seedlings with roots into nutrient soil to obtain candidate transgenic seedlings of pc186-TaHRF1 and pc186-GUS respectively.

[0103] Table 1: Composition of culture medium for wheat genetic transformation

[0104]

[0105] 2. PCR detection of candidate transgenic plants

[0106] The genomic DNA of T0 generation candidate wheat plants transfected with pc186-TaHRF1 vector and pc186-GUS vector was extracted using the CTAB method (Sambrook and Russell, Molecular Cloning Laboratory Guide, 2001).

[0107] The extracted genomic DNA of the candidate transgenic plant was used as a template, and primers (upstream primer: 5'-GGCGGTCTGCACCATCGTCAACCACTAC-3', SEQ ID NO.9; downstream primer: 5'-AGTCCAGCTGCCAGAAACCCACGTCATG-3', SEQ ID NO.10) were designed for PCR amplification to detect the presence of the bar gene. If the bar gene is present, the length of the amplified fragment is 446 bp. The total volume of the amplification system is 20 μl, including 1 μl upstream primer (10 μmol / μl), 1 μl downstream primer (10 μmol / μl), 10 μl 2×Rapid Taq Master Mix, 1 μl genomic DNA template (100-200 ng / μl) and 7 μl ddH2O. Amplification conditions were as follows: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, extension at 72°C for 15 seconds, denaturation, annealing, and extension for 32 cycles; and extension at 72°C for 5 minutes.

[0108] PCR identification results Figure 5 As shown, there is no bar gene fragment with a length of 446 bp in wild-type wheat.

[0109] 3. Statistics of transformation efficiency of different wheat genotypes

[0110] After wheat young embryos were infected with Agrobacterium 14-15 days after pollination, the callus induced was screened, and the number of induced callus was counted when it was transferred to LSF-P5 medium; after PCR identification, the number of transgenic positive seedlings was counted, and finally the callus induction rate and transformation efficiency were calculated. The calculation formula is as follows:

[0111] Callus induction rate (%) = (callus number ÷ total number of young embryos) × 100%;

[0112] Transformation efficiency (%) = (number of positive seedlings ÷ total number of young embryos) × 100%;

[0113] Compared with the control vector pc186-GUS, transformation with the pc186-TaHRF1 vector can effectively improve the transformation efficiency of wheat. The results are shown in Table 1.

[0114] Table 2: Comparison of transformation efficiency between control vector pc186-GUS and pc186-TaHRF1 vector

[0115]

[0116] The immature embryos of the wheat production promotion variety Liangxing 66 were used as explants. After the Agrobacterium-mediated transformation of the pc186-TaHRF1 vector and the control vector pc186-GUS, the callus induction rates were 95.27% and 89.63%, respectively. The transformation efficiency of the pc186-TaHRF1 vector was 45.56%, while the control vector could not obtain transgenic plants. When the backbone wheat variety Aifeng No. 3 was used as the recipient for genetic transformation, the control vector pc186-GUS failed to obtain positive transgenic plants, while the transformation efficiency of the pc186-TaHRF1 vector was 39.84%. From the above results, it can be seen that the TaHRF1 gene can greatly improve the transformation efficiency of wheat and can solve the genotype dependence problem in some wheat genetic transformation.

[0117] IV. Phenotypic investigation of transgenic plants:

[0118] The growth and development of the positive plants obtained by transforming Fielder with the plant expression vector pc186-TaHRF1 and the wild-type Fielder were investigated. Figure 6 As shown, the results showed that plants overexpressing the TaHRF1 gene grew and developed normally and did not affect the agronomic traits of wheat.

[0119] 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 TaHRF1 gene in the following (1) or (2): (1) Improve the transformation efficiency of nucleic acid molecules into the wheat genome; (2) Stable expression of nucleic acid molecules improves the regeneration efficiency of wheat plants.

2. The use according to claim 1, characterized in that: The TaHRF1 gene is a nucleic acid molecule as shown in the following i) or ii) or iii): i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) nucleic acid molecules that have 80% or more homology with the nucleotide sequence of i) and express proteins with the same functions, as well as corresponding alleles, homologous genes, mutant genes and derivative genes; iii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

3. Use of the protein encoded by the TaHRF1 gene in the following (1) or (2): (1) Improve the transformation efficiency of nucleic acid molecules into the wheat genome; (2) Stable expression of nucleic acid molecules improves the regeneration efficiency of wheat plants.

4. The use according to claim 3, characterized in that: The protein encoded by the TaHRF1 gene is a protein as shown in any one of (A1) or (A2) or (A3) 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). (A3) The encoded protein is similar to the protein shown in SEQ ID NO. 2, or a protein obtained by substitution, deletion or insertion of one, several or dozens of amino acids.

5. Use of an expression cassette, a recombinant expression vector or a recombinant bacterium containing the TaHRF1 gene in the following (1) or (2): (1) Improve the transformation efficiency of nucleic acid molecules into the wheat genome; (2) Stable expression of nucleic acid molecules improves the regeneration efficiency of wheat plants.

6. A method for improving wheat genetic transformation efficiency, characterized in that: The following steps are involved: The TaHRF1 gene and the target nucleic acid molecule are transferred into the target plant to achieve the purpose of improving the transformation efficiency of the nucleic acid molecule introduced into the target plant; The TaHRF1 gene is a nucleic acid molecule as shown in the following i) or ii) or iii): i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) nucleic acid molecules that have 80% or more homology with the nucleotide sequence of i) and express proteins with the same functions, as well as corresponding alleles, homologous genes, mutant genes and derivative genes; iii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

7. The method according to claim 6, characterized in that The TaHRF1 gene and the target nucleic acid molecule are transferred into the target plant via one vector or via different vectors.

8. The method according to claim 7, characterized in that The TaHRF1 gene and target nucleic acid molecule are transferred into the target plant via the pc186 expression vector.

9. The method according to claim 6, characterized in that The target plant is a grass plant.

10. The method according to claim 9, characterized in that The target plant is wheat.

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