Application of TaRF1 gene and its encoded protein in improving wheat transformation efficiency

Abstract

The present invention discloses the application of the TaRF1 gene and the protein it encodes in improving the transformation efficiency of wheat, and belongs to the technical field of plant genetic engineering. The TaRF1 gene can improve the transformation efficiency of nucleic acid molecules introduced into target plants and / or promote the introduction of nucleic acid molecules into target plants, and the target plants include but are not limited to monocotyledons such as wheat. An overexpression vector is constructed using the CDS sequence of the TaRF1 gene, and the TaRF1 gene overexpression vector is introduced into an Agrobacterium strain, and the wheat embryos are infected by the Agrobacterium-mediated method. The results showed that compared with the control vector, the TaRF1 gene overexpression vector can promote the entry of nucleic acid molecules into the target plant. The use of the TaRF1 gene can improve the transformation efficiency of the target gene introduced into the plant, improve the genetic transformation efficiency of monocotyledons, especially wheat, and has important economic value and social benefits for the study of plant gene function and the improvement of crop agronomic traits.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to the application of TaRF1 gene and the protein encoded therein in improving wheat transformation efficiency. Background Art

[0002] Wheat (Triticum aestivum L.) is a vital food crop, providing food for one-third of the world's population. Global food security is an increasingly prominent issue, crucial for economic development and social stability. With the continuous advancement of biotechnology, the use of genetic engineering to improve wheat's agronomic traits, such as yield and quality, and to enhance its resistance to pests, diseases, and other biotic and abiotic stresses has become a significant trend. After years of continuous research and effort, genetic engineering breeding has gradually become an important supplement to conventional wheat breeding (Wan Jianmin, Life Sciences, 2011).

[0003] As an allohexaploid plant, wheat has a large genome, numerous repetitive sequences, and the vast majority of genotypes have poor regeneration capacity, making it a major crop that is difficult to genetically transform. Wheat's efficient regeneration capacity is the foundation of genetic transformation, but genotypes with high regeneration capacity are relatively rare, and most are not commercially available varieties. This strong genotype dependency is a major factor limiting wheat genetic transformation. Although wheat explants, including immature embryos, mature embryos, anthers, young ears, leaves, and growing points, can be regenerated into complete plants through in vitro culture, immature embryos have the strongest regeneration capacity. Consequently, most wheat transgenic research has utilized immature embryo explants, accounting for at least 90% of reports, resulting in a significant explant dependency (Ye Xingguo et al., Chinese Journal of Agricultural Sciences, 2014). These issues have limited the development of wheat gene function research and become a bottleneck in the development of wheat biotechnology breeding.

[0004] Currently, research on genes that improve wheat transformation efficiency is relatively limited. Patent CN 108997484A cloned the wheat TaWOX5 gene and found that introducing this gene into different wheat varieties significantly improved transformation efficiency. Patent CN112521475A cloned the TaLAX1-A gene from wheat. Overexpressing this gene and applying it to key germplasm resources with regeneration difficulties can improve genetic transformation efficiency. Therefore, further isolation of new genes that can improve wheat genetic transformation efficiency is of great significance for improving crop agronomic traits, enhancing wheat's resistance to pests and diseases, and to biotic and abiotic stresses, and promoting the industrialization of transgenic wheat. Summary of the Invention

[0005] In view of the above-mentioned prior art, the object of the present invention is to provide an application of the TaRF1 gene and the protein encoded by the gene in improving the transformation efficiency of wheat.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides the use of the TaRF1 gene in the following (1) or (2):

[0008] (1) Improve the transformation efficiency of nucleic acid molecules into wheat;

[0009] (2) Improve the efficiency of wheat embryo regeneration;

[0010] The TaRF1 gene is a nucleic acid molecule as shown in the following i) or ii) or iii):

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

[0012] 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;

[0013] iii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 2 except i).

[0014] 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.

[0015] The term "homology" as used herein refers to sequence similarity to a natural nucleic acid sequence. Homology can be evaluated using computer software, for example, the BLAST algorithm (Altschul et al. 1990. Journal of Molecular Biology 215: 403-410; Karlin and Altschul. 1993. Proceedings of the National Academy of Sciences 90: 5873-5877).

[0016] In the above nucleic acid molecules, the 80% or more homology may be at least 80%, 85%, 90%, 95%, 96%, 98% or 99% homology.

[0017] The second aspect of the present invention provides the use of a protein encoded by the TaRF1 gene in the following (1) or (2):

[0018] (1) Improve the transformation efficiency of nucleic acid molecules into wheat;

[0019] (2) Improve the efficiency of wheat embryo regeneration.

[0020] Preferably, the protein encoded by the TaRF1 gene is a protein as shown in any one of the following (A1) or (A2) or (A3):

[0021] (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO. 2 in the sequence listing;

[0022] (A2) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0023] (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.

[0024] The proteins described in (A1), (A2) and (A3) can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0025] In the above proteins, a protein tag refers to a polypeptide or protein that is fused and expressed with a target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. To facilitate purification of the protein in (A1), a tag may be attached to the amino or carboxyl terminus of the protein in (A1). The tag may be Poly-Arg (usually six RRRRRs), Poly-His (usually six HHHHHHs), FLAG (DYKDDDDK), Strep-tag II (WSHPQFEK), or c-Myc (EQKLISEEDL).

[0026] The third aspect of the present invention provides the use of an expression cassette, a recombinant expression vector or a recombinant bacterium containing the TaRF1 gene in the following (1) or (2):

[0027] (1) Improve the transformation efficiency of nucleic acid molecules into wheat;

[0028] (2) Improve the efficiency of wheat embryo regeneration.

[0029] The recombinant expression vector can be constructed using existing plant expression vectors. Preferably, the recombinant expression vector containing the TaRF1 gene is constructed using the pc186 expression vector.

[0030] A fourth aspect of the present invention provides a method for improving the transformation efficiency of a nucleic acid molecule into a target plant, comprising the following steps:

[0031] The TaRF1 gene and nucleic acid molecule are transferred into the target plant to achieve the purpose of improving the transformation efficiency of the nucleic acid molecule into the target plant;

[0032] The TaRF1 gene is a nucleic acid molecule as shown in the following i) or ii) or iii):

[0033] i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1;

[0034] 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;

[0035] iii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 2 except i).

[0036] In the above method, the TaRF1 gene and the nucleic acid molecule can be introduced into the target plant via one vector or via different vectors.

[0037] Preferably, the TaRF1 gene and nucleic acid molecule are introduced into the target plant via the pc186 expression vector.

[0038] 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.

[0039] Beneficial effects of the present invention:

[0040] The present invention discovered for the first time that the TaRF1 gene can improve the transformation efficiency of nucleic acid molecules introduced into target plants and / or promote the introduction of nucleic acid molecules into target plants, and the target plants include but are not limited to monocotyledonous plants such as wheat. An overexpression vector was constructed using the CDS sequence of the TaRF1 gene, and it was introduced into an Agrobacterium strain, and the wheat embryos were infected by the Agrobacterium-mediated method. The results showed that compared with the control vector, the TaRF1 gene overexpression vector can promote the entry of nucleic acid molecules into the target plant. The use of the TaRF1 gene can improve the transformation efficiency of the target gene introduced into plants, improve the genetic transformation efficiency of monocotyledonous plants, especially wheat, and has important economic value and social benefits for the study of plant gene function and the improvement of crop agronomic traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a schematic diagram of the partial structure of the plant expression vector pc186-TaRF1;

[0042] Figure 2 This is a schematic diagram of the partial structure of the plant expression vector pc186-GUS vector;

[0043] Figure 3Schematic diagram of PCR-specific amplification of the bar gene in candidate transgenic plants transformed with the plant expression vector pc186-TaRF1. In the figure, M represents a 2000 bp molecular weight marker, PC represents a positive plasmid, CK represents a wild-type control, and L1-L10 represent candidate transgenic plants. DETAILED DESCRIPTION

[0044] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0045] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0046] The unspecified test materials used in the examples of the present invention are all conventional test materials in this area and can be purchased through commercial channels. The present invention introduces the expression vector into the plant cell, and the introduction methods are all well known to those skilled in the art, including but not limited to: Agrobacterium-mediated method, gene gun bombardment method, electroporation method, ovary injection method, etc. The selective marker gene used in the present invention is the bar gene, which encodes the phosphinothricin acetyltransferase PAT protein. Other selective marker genes and reporter genes including nptII and hpt can be further used. The screening antibiotic selected by the present invention is phosphinothricin, and the selection of screening agents such as bialaphos can also have the same effect. Where specific experimental conditions and methods are not specified in the examples of the present invention, conventional conditions are generally used, such as J. Sambrook et al., ed., Science Press, 2002, Molecular Cloning Experiment Guide (3rd edition); DL Spector et al., ed., Science Press, 2001, Cell Experiment Guide; or according to the conditions recommended by the manufacturer.

[0047] Example 1: Cloning of TaRF1 gene and construction of expression vector

[0048] Total RNA from the wheat line Fielder was extracted using the Ultrapure RNA Kit (Chemical World, Cat. No. CW0581M).

[0049] cDNA was reverse transcribed using the FastKing RT Kit (With gDNase) (Tiangen Biochemical Technology (Beijing) Co., Ltd., Cat. No. KR116).

[0050] PCR amplification was performed using the cDNA as a template using a primer pair (upstream primer: 5'-ATGGCTCCAGCCTCCGC-3', SEQ ID NO. 3; downstream primer: 5'-TCAGTTGCCGGCCTCCG-3', SEQ ID NO. 4). The amplification system consisted of 2 μl of upstream primer (10 μmol / μl), 2 μl of downstream primer (10 μmol / μl), 12.5 μl of 2× Phanta Max Master Mix, 1 μl of cDNA template, and ddH2O to a total volume of 25 μl. Amplification conditions were: initial denaturation at 95°C for 3 minutes, followed by 35 cycles of denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, and extension at 72°C for 30 seconds, followed by extension at 72°C for 5 minutes.

[0051] The amplified PCR products were -Blunt3 Cloning Kit (Beijing Quanshijin Biotechnology Co., Ltd., Cat. No. CB301-01) Operation steps and connection -Blunt3 vector to obtain pEASY-Blunt3-TaRF1 vector, and sequenced it.

[0052] After sequencing analysis, the nucleotide sequence of the PCR amplification product is Sequence 1 in the sequence listing. The gene represented by the PCR product is named TaRF1 gene; the protein encoded by the gene is named TaRF1, and the amino acid sequence of the protein is Sequence 2 in the sequence listing. The details are as follows:

[0053] Sequence 1 (SEQ ID NO.1):

[0054] ATGGCTCCAGCCTCCGCCGGCGCCAAGCGCCCCTTCGCCGCCGACTCCGCCCACGACGCTGCTGAGCAAGAGCAATCACTCGCGCAGGTCCAGGAGAGCGCGGTGAGCAAGAAGAACGGCCAGAGCGAGCAGCCGAAGCTGGAGTGCCCACGGTGCAGCTCCACGGACACCAAGTTCTGCTACTACAACAACTACAGCACGGCACAGCCGCGCCACTACTGCCGCACCTGCCGCCGCTACTGGACGCACGGCGGCACGCTGCGCAAGGTCCCCGTCGGCGGCGCCTGCCGCCGCGGCTCCGGCAGCAGCAGCAAGCGCCGCAGGCCCTCCGCCGAGCCCCACACGCCCTCCTCCGGCTCGCCGCAGCCGGACCAGCAGGAGCAAGACACGCTCCCCCCGCTCCCGGTCTTCCCGTTCCTCACCGATGGCGGCCCCGCCTTCCTGCCGCAGTTCGACCTCGGGCTCTACGACGGGCTCGCGGCGCCCACCGGCGCATGGGAGGACTTCGGCGGCCTTGAGCTCACCTGGCCACCACCGCCACCCCCGGAGGCCGGCAACTGA

[0055] Sequence 2 (SEQ ID NO.2):

[0056] MAPASAGAKRPFAADSAHDAAEQEQSLAQVQESAVSKKNGQSEQPKLECPRCSSTDTKFCYYNNYSTAQPRHYCRTCRRYWTHGGTLRKVPVGGACRRGSGSSSKRRRPSAEPHTPSSGSPQPDQQEQDTLPPLPVFPFLTDGGPAFLPQFDLGLYDGLAAPTGAWEDFGGLELTWPPPPPPEAGN

[0057] Sequence 1 can also be synthesized manually and ligated to the pEASY-Blunt3 vector to obtain the pEASY-Blunt3-TaRF1 vector.

[0058] Using pEASY-Blunt3-TaRF1 as a template, a primer pair (upstream primer: 5'-CACCATGGCTCCAGCCTCCGC-3', SEQ ID NO. 5; downstream primer: 5'-TCAGTTGCCGGCCTCCG-3', SEQ ID NO. 4) was designed for PCR amplification. The amplification system consisted of 2 μl of upstream primer (10 μmol / μl), 2 μl of downstream primer (10 μmol / μl), 12.5 μl of 2× Phanta Max Master Mix, 1 μl of cDNA template, and ddH2O to a total volume of 25 μl. Amplification conditions were: initial denaturation at 95°C for 3 minutes, followed by 35 cycles of denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, and extension at 72°C for 30 seconds, followed by extension at 72°C for 5 minutes.

[0059] The amplified PCR product was referred to pENTR TM Directional Cloning Kit (ThermoScientific TM , Catalog No.: K2400-20SP) Operation steps for ligation sequencing. The correctly sequenced single clone was ligated into the pc186 expression vector through LR reaction to obtain the pc186-TaRF1 vector. The schematic diagram of the vector structure is shown in the figure. Figure 1 shown.

[0060] pc186-TaRF1 was transformed into Agrobacterium EHA105 competent cells, and an Agrobacterium strain suitable for transformation was obtained, named pc186-TaRF1 / EHA105.

[0061] Example 2: Construction of control vector pc186-GUS

[0062] Refer to NCBI ( https: / / www.ncbi.nlm.nih.gov / PCR amplification was performed using nucleotides 15108-16919 of Sequence ID: MN266288.1 on the website. The primer pair (upstream primer: 5'-ATGTTACGTCCTGTAGAA-3', SEQ ID NO. 6; downstream primer: 5'-TCATTGTTTGCCTCCCTG-3', SEQ ID NO. 7) was used. The amplification system consisted of 2 μl of upstream primer (10 μmol / μl), 2 μl of downstream primer (10 μmol / μl), 12.5 μl of 2× Phanta Max Master Mix, 1 μl of cDNA template, and ddH2O added to bring the total volume to 25 μl. Amplification conditions were: initial denaturation at 95°C for 3 minutes, followed by 35 cycles of denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, and extension at 72°C for 55 seconds, followed by extension at 72°C for 5 minutes.

[0063] The amplified PCR products were -Blunt3 Cloning Kit (Cat. No. CB301-01, Beijing Quanshijin Biotechnology Co., Ltd.) was used to connect the pEASY-B3-GUS, which was then sequenced.

[0064] After sequencing analysis, the gene indicated by the PCR product was named GUS gene.

[0065] The GUS gene can also be artificially synthesized and connected to the pEASY-Blunt3 vector to obtain pEASY-Blunt3-GUS.

[0066] Using pEASY-Blunt3-GUS as a template, a primer pair (upstream primer: 5'-CACCATGTTACGTCCTGTAGAA-3', SEQ ID NO. 8; downstream primer: 5'-TCATTGTTTGCCTCCCTG-3', SEQ ID NO. 7) was designed for PCR amplification. The amplification system consisted of 2 μl of upstream primer (10 μmol / μl), 2 μl of downstream primer (10 μmol / μl), 12.5 μl of 2× Phanta Max Master Mix, 1 μl of cDNA template, and ddH2O to a total volume of 25 μl. Amplification conditions were: initial denaturation at 95°C for 3 minutes, followed by 35 cycles of denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, and extension at 72°C for 55 seconds, followed by extension at 72°C for 5 minutes.

[0067] 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 correct sequenced single clone was ligated into the pc186 expression vector by LR reaction to obtain the pc186-GUS vector. The schematic diagram of the vector structure is shown in Figure 2. Figure 2 shown.

[0068] pc186-GUS was transformed into Agrobacterium EHA105 competent cells, and an Agrobacterium strain suitable for transformation was obtained, named pc186-GUS / EHA105.

[0069] Example 3: Agrobacterium-mediated transformation of wheat immature embryos and identification of resistant plants

[0070] 1. For 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:

[0071] 1. Three days before infection, inoculate pc186-TaRF1 / EHA105 and pc186-GUS / EHA105 Agrobacterium onto solid YEP medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, respectively. Incubate in the dark at 28°C for 2 days. Pick a single colony and inoculate it into liquid YEP medium containing 50 mg / L kanamycin and 50 mg / L rifampicin. Incubate with shaking at 220 rpm at 28°C overnight. Pour the above Agrobacterium solution into a 2 ml centrifuge tube, centrifuge at 6000 rpm for 5 minutes, discard the supernatant, and resuspend the pellet with resuspension buffer to obtain Agrobacterium resuspensions of pc186-TaRF1 / EHA105 and pc186-GUS / EHA105, respectively.

[0072] 2. Approximately 14 days after flowering, immature embryos of different wheat genotypes were infected with Agrobacterium resuspensions of pc186-TaRF1 / EHA105 and pc186-GUS / EHA105, respectively, and spread with the scutellum side up on WLS-AS medium (1 / 10MS minimal medium, 1 / 10MS vitamins, 10 g / L glucose, 100 μM acetosyringone, 8 g / L agarose), and incubated in a dark incubator at 23°C for 2 days.

[0073] 3. After co-cultivation, the immature embryos were transferred to WLS-Res medium (MS minimal medium, MS vitamins, 2,4-D 0.5 mg / L, picloram 2.2 mg / L, glutamine 0.5 g / L, casein 0.1 g / L, MgCl2·6H2O 0.75 g / L, maltose 40 g / L, AgNO3 0.85 mg / L, vitamin C 100 mg / L, carbenicillin 250 mg / L, agarose 5 g / L) and cultured in a dark incubator at 25°C for 5 days.

[0074] 4. Transfer the callus tissue after recovery culture to WLS-P5 medium (WLS-Res medium supplemented with PPT 5 mg / L) and culture in a dark incubator at 25°C for 14 days.

[0075] 5. The callus tissue was then transferred to WLS-P10 medium (WLS-Res medium supplemented with PPT 10 mg / L) and cultured in a 25°C incubator in the dark for 21 days.

[0076] 6. The callus was transferred to LSZ-P5 medium (MS basic medium, LS vitamins, zeatin 5 mg / L, sucrose 20 g / L, carbenicillin 250 mg / L, PPT 5 mg / L, phytagel 3 g / L), and cultured in a 25°C incubator under light for 2 weeks.

[0077] 7. Transfer the regenerated buds of wheat callus to LSF-P5 medium (MS basic medium, LS vitamins, IBA0.2 mg / L, sucrose 15 g / L, carbenicillin 250 mg / L, PPT 5 mg / L, phytogel 3 g / L) and culture in a 25°C incubator under light until the roots of the regenerated buds are about 1-2 cm long.

[0078] 8. Transplant the rooted seedlings into nutrient soil to obtain resistant seedlings of pc186-TaRF1 and pc186-GUS respectively.

[0079] 2. PCR detection of candidate transgenic plants

[0080] The CTAB method (Sambrook and Russell, Molecular Cloning Laboratory Manual, 2001) was used to extract genomic DNA from leaves of T0 generation wheat plants transfected with pc186-TaRF1 and pc186-GUS vectors.

[0081] Primers were designed to detect the bar gene. The primer pair sequence (upstream primer: 5'-GGCGGTCTGCACCATCGTCAACCACTAC-3', SEQ ID NO. 9; downstream primer: 5'-AGTCCAGCTGCCAGAAACCCACGTCATG-3', SEQ ID NO. 10) amplified a 446-bp sequence. The amplification system consisted of 1 μl of the upstream primer (10 μmol / μl), 1 μl of the downstream primer (10 μmol / μl), 10 μl of 2× Rapid Taq Master Mix, and 1 μl of cDNA template. The total volume was adjusted to 20 μl with ddH2O. Amplification conditions were: initial denaturation at 95°C for 3 minutes, followed by 32 cycles of denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, and extension at 72°C for 15 seconds, followed by extension at 72°C for 5 minutes.

[0082] PCR identification results Figure 3 As shown, there is no 446 bp bar gene fragment in wild-type wheat.

[0083] 3. Transformation efficiency statistics of different wheat genotypes

[0084] After wheat embryos were infected with Agrobacterium, the calli induced were screened and the number of resistant calli formed was counted when they were transferred to LSF-P5 medium. After PCR identification, the number of positive seedlings was counted, and the resistant callus induction rate and transformation efficiency were finally calculated using the following formula:

[0085] Resistant callus induction rate (%) = (number of resistant calli ÷ total number of immature embryos) × 100%;

[0086] Transformation efficiency (%) = (number of positive seedlings ÷ total number of embryos) × 100%;

[0087] Compared with the control vector pc186-GUS, transformation with the pc186-TaRF1 vector can effectively improve the transformation efficiency of wheat. The results are shown in Table 1.

[0088] Table 1: Comparison of transformation efficiency between control vector and pc186-TaRF1 vector

[0089]

[0090] The induction rate and transformation efficiency of immature embryo explants of the wheat line Fielder transformed with the pc186-TaRF1 vector were 94.38% and 164.38%, respectively. These figures were significantly higher than the 92.98% induction rate and 32.46% transformation efficiency of the control vector pc186-GUS. In genetic transformation of Jimai 22, the control vector pc186-GUS failed to produce positive transgenic plants, while the pc186-TaRF1 vector achieved a transformation efficiency of 33.33%. These results demonstrate that the TaRF1 gene can significantly improve transformation efficiency in wheat and partially address the genotype-dependent nature of wheat transformation.

[0091] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. Overexpression TaRF1 The use of genes in the following (1) or (2): (1) Improve the transformation efficiency of nucleic acid molecules into wheat; (2) Improve the efficiency of wheat embryo regeneration; described TaRF1 A gene is a nucleic acid molecule as shown in i) or ii) below: i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 2 except i).

2. Overexpression TaRF1 Application of the protein encoded by the gene in the following (1) or (2): (1) Improve the transformation efficiency of nucleic acid molecules into wheat; (2) Improve the efficiency of wheat embryo regeneration; described TaRF1 The protein encoded by the gene is as shown below (A1) or (A2): (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO. 2 in the sequence listing; (A2) Fusion proteins obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

3. Contains TaRF1 Application of gene expression cassettes, recombinant expression vectors or recombinant bacteria in the following (1) or (2): (1) Improve the transformation efficiency of nucleic acid molecules into wheat; (2) Improve the efficiency of wheat embryo regeneration; described TaRF1 A gene is a nucleic acid molecule as shown in i) or ii) below: i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 2 except i).

4. A method for improving the transformation efficiency of nucleic acid molecules into target plants, characterized in that: The following steps are involved: Will TaRF1 Genes and nucleic acid molecules are transferred into target plants to achieve the purpose of improving the transformation efficiency of nucleic acid molecules into target plants; described TaRF1 A gene is a nucleic acid molecule as shown in i) or ii) below: i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 2 other than i); The target plant is wheat.

5. The method according to claim 4, characterized in that TaRF1 Genes and nucleic acid molecules are transferred into target plants via one vector or via different vectors.

6. The method according to claim 5, characterized in that TaRF1 The genes and nucleic acid molecules are transferred into target plants through the pc186 expression vector.

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