A gene for improving the efficiency of genetic transformation of wheat and application thereof

Abstract

The application discloses a gene for improving wheat genetic transformation efficiency and application thereof, and belongs to the technical field of plant genetic engineering. The TaHRF1 gene and the coded protein thereof can promote integration of an exogenous nucleic acid molecule into a target plant genome, improve nucleic acid molecule integration efficiency, and make a transformed plant grow normally, and the target plant includes but is not limited to monocotyledonous plants such as wheat. A wheat TaHRF1 gene overexpression vector is constructed to infect explants of different genotypes of wheat. Statistics show that, compared with a control vector, the TaHRF1 gene overexpression vector can promote integration of a nucleic acid molecule into a target plant genome. The TaHRF1 gene can improve genetic transformation efficiency of a target gene into a plant, improve regeneration ability and transformation efficiency of monocotyledonous plants, especially wheat, and make a genetically transformed plant grow normally. The application provides a new important gene resource for overcoming genotype dependence of wheat genetic transformation.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a gene that improves the efficiency of wheat genetic transformation and its application. Background Technology

[0002] Wheat is a vital food crop worldwide, and its high yield, quality, stability, and efficiency are closely related to food security, socio-economic stability, and human nutrition and health. Wheat breeding is a major technological means to achieve high yield, quality, stability, and efficiency in wheat. Genetic transformation technology and gene editing are important techniques for improving agronomic traits such as crop yield, disease resistance, and quality, and have already 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, and poor regeneration capacity, making genetic transformation more difficult. This has become the main reason for the slow progress in wheat bio-breeding.

[0003] Researchers have explored various transformation methods to introduce exogenous genes into target plants, including gene gun methods, Agrobacterium-mediated transformation, pollen tube pathway methods, ultrasound methods, ion beam implantation, laser microbeam puncture, and PEG (Polyethylene glycol) methods. Agrobacterium-mediated transformation is currently the most commonly used method for wheat transformation, primarily using Agrobacterium tumefaciens. This method offers advantages such as low cost, simple operation, high efficiency in introducing exogenous genes, low copy number, and good genetic stability. Gene gun methods are physical transformation methods that introduce exogenous genes into organisms through high-speed particle bombardment. They offer advantages such as genotype independence and direct transformation, and are also one of the commonly used transformation methods.

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

[0005] In recent years, significant breakthroughs have been made in wheat genetic transformation technology. Overexpression of key genes that promote regeneration can improve crop regeneration capacity and genetic transformation efficiency, and this new method for enhancing wheat genetic transformation efficiency is gradually gaining acceptance. However, the number of regeneration-related genes isolated so far is still relatively 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 this invention is to provide a gene for improving the genetic transformation efficiency of wheat and its application. This invention has found that overexpression of the wheat TaHRF1 gene significantly improves the transformation efficiency of multiple wheat varieties, effectively overcoming the genotype dependence of wheat. The application of TaHRF1 can improve the genetic transformation efficiency of plants and can be used as a transformation helper gene to improve the genetic transformation efficiency of crops such as wheat, reduce genetic transformation costs, and lay a solid foundation for biotechnology breeding.

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

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

[0009] (1) Improve the conversion efficiency of nucleic acid molecules integrating 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 i), 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 i) in nucleotide sequence and express the same or similar functional proteins, as well as the corresponding alleles, homologous genes, mutant genes and derived genes;

[0014] iii) Nucleic acid molecules other than i) encoding the amino acid sequence shown in SEQ ID NO.2.

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

[0016] The term "homology" used here refers to sequence similarity to natural nucleic acid sequences. Homology can be evaluated using computer software, such as the BLAST algorithm (Altschulet 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 aforementioned nucleic acid molecules, the 80% or more homology can be at least 80%, 85%, 90%, 95%, 96%, 98%, or 99% homology.

[0018] A second aspect of the invention provides the use of the protein encoded by the TaHRF1 gene in either (1) or (2) below:

[0019] (1) Improve the conversion efficiency of nucleic acid molecules integrating 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 any one of the following proteins: (A1), (A2), or (A3):

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

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

[0024] (A3) encodes a protein 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 aforementioned proteins, a protein tag refers to a polypeptide or protein expressed fused with the target protein using in vitro DNA recombination technology, facilitating the expression, detection, tracing, and / or purification of the target protein. Specifically, to facilitate the purification of the protein in (A1), a tag can be attached to the amino or carboxyl terminus of the protein in (A1). The tag can be Poly-Arg (typically 6 RRRRR), Poly-His (typically 6 HHHHHH), FLAG (DYKDDDDK), Strep-tagII (WSHPQFEK), or c-Myc (EQKLISEEDL).

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

[0028] (1) Improve the conversion efficiency of nucleic acid molecules integrating 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, a 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 nucleic acid molecules introduced into target plants, comprising the following steps:

[0032] The TaHRF1 gene and target nucleic acid molecules are transferred into target plants to improve the transformation efficiency of target nucleic acid molecules in target plants.

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

[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 i) in nucleotide sequence and express the same or similar functional proteins, as well as the corresponding alleles, homologous genes, mutant genes and derived genes;

[0036] iii) Nucleic acid molecules other than i) encoding the amino acid sequence shown in SEQ ID NO.2.

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

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

[0039] In the above method, the target plant includes, but is 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] The beneficial effects of this invention are:

[0041] This invention is the first to discover that the TaHRF1 gene can improve the efficiency of target nucleic acid molecule integration into target plants and / or promote the integration of nucleic acid molecules into target plants, including but not limited to monocotyledonous plants such as wheat. An overexpression vector was constructed using the CDS sequence of the TaHRF1 gene, introduced into Agrobacterium strains, and infected with immature embryos of different wheat genotypes. The results showed that, compared with the control vector, the TaHRF1 gene overexpression vector promoted the entry and integration of target nucleic acid molecules into the genome of the target plants. The TaHRF1 gene can improve the efficiency of target gene integration into the plant genome, increase the genetic transformation efficiency of monocotyledonous plants, especially wheat, and the genetically transformed plants exhibit normal growth and development. This invention has significant economic and social value for accelerating plant gene function research and improving crop agronomic traits. Attached Figure Description

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

[0043] Figure 2 A schematic diagram of the structure of the plant expression vector pc186-GUS.

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

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

[0046] Figure 5This diagram illustrates 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 diagram, 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 a 2000bp molecular weight marker.

[0047] Figure 6 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 Implementation

[0048] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0049] As mentioned earlier, due to the large and complex genome of wheat, the high number of repetitive DNA sequences, and its low regeneration capacity, the application of genetic transformation and gene editing technologies in wheat lags far behind that of other major crops. Previous studies have found that regeneration genes can improve the regeneration efficiency and genetic transformation efficiency of wheat plants in in vitro tissue culture; however, the number of wheat regeneration genes isolated and identified is still very small. The transformation efficiency varies significantly among different wheat genotypes, and most wheat genotypes cannot be successfully transformed. Furthermore, the introduction of exogenous wheat regeneration genes may have adverse effects on wheat agronomic traits. Therefore, identifying wheat regeneration genes with practical production application value is very difficult.

[0050] "Liangxing 66" is a wheat variety bred by Shandong Liangxing Seed Industry Co., Ltd. using varieties Ji 91102 / Ji 935031. It was approved by the Fourth Meeting of the Second National Crop Variety Approval Committee on December 13, 2010, with the approval number Guoshenmai 2010004. This variety is characterized by high yield, good quality, high lodging resistance, high productivity, and wide suitability for various regions. "Aifeng 3" is a wheat variety bred under the leadership of Academician Zhao Hongzhang of Northwest A&F University. It is the first semi-dwarf variety to be widely promoted in the history of wheat production in my country. Both wheat varieties have been on the market for many years and now face the challenge of further genetic improvement.

[0051] However, "Liangxing 66" and "Aifeng 3" are wheat varieties that are extremely difficult to transform, making genetic integration and gene editing of them quite challenging.

[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 transferring the TaHRF1 gene from wheat into the wheat varieties “Liangxing 66” and “Aifeng 3” using Agrobacterium-mediated transformation can significantly improve the genetic transformation efficiency of these two extremely difficult-to-transform wheat varieties.

[0053] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0054] The experimental materials used in the embodiments of this invention, unless otherwise specified, are all conventional experimental materials in the art and can be purchased through commercial channels. This invention introduces the expression vector into plant cells using methods well-known to those skilled in the art, including but not limited to: Agrobacterium-mediated transformation, gene gun bombardment, electroporation, ovary injection, etc. The selectable marker gene used in this invention is the bar gene, encoding glufosinate acetyltransferase PAT protein; other selectable marker genes and reporter genes, including nptII and hpt, can be further used. The screening antibiotic used in this invention is glufosinate; screening agents such as bialaphos can also achieve the same effect. Where specific experimental conditions and methods are not specified in the embodiments of this invention, they are generally performed according to conventional conditions, such as those described in J. Sambrook et al., eds., *Molecular Cloning: A Laboratory Manual* (3rd edition), Science Press, 2002; and D.L. Spector et al., eds., *Cellular Laboratory Manual*, Science Press, 2001; or according to the manufacturer's recommendations.

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

[0056] Callus tissue was collected from wheat Fielder embryo explants after tissue culture induction. Total RNA was first extracted using the Ultrapure RNA Kit (Kangwei Century, catalog number: CW0581M), and then the extracted RNA was reverse transcribed into cDNA using the FastKing RT Kit (With gDNase) (Tiangen Biotech (Beijing) Co., Ltd., catalog number: KR116).

[0057] cDNA was amplified using 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 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 DNA template, and 7.5 μl of ddH2O. The amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 30 s, for 32 cycles; 72℃ extension for 5 min; and final hold at 16℃.

[0058] Reference -Blunt3 Cloning Kit (Beijing TransGen Biotech Co., Ltd., Catalog No.: CB301-01) Operation Steps: Ligate PCR amplification products to... The -Blunt3 vector was used to obtain the pEASY-Blunt3-TaHRF1 vector, and the results were analyzed after Sanger sequencing.

[0059] Sequencing analysis revealed that the nucleotide sequence of the PCR amplification product corresponds to sequence 1 in the sequence listing. The gene represented by this PCR product is named TaHRF1; the protein encoded by this gene is named TaHRF1, and its amino acid sequence corresponds to sequence 2 in the sequence listing. 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] Alternatively, sequence 1 can be artificially synthesized and linked to the pEASY-Blunt3 vector to obtain the pEASY-Blunt3-TaHRF1 vector.

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

[0083] Refer to pENTR TM Directional Cloning Kit (Thermo Scientific) TM (Catalog No.: K2400-20SP) Procedure: Directly clone the amplified PCR product into the Gateway system, followed by sequencing. Successfully sequenced single clones are ligated into the pc186 expression vector via the LR reaction to obtain the pc186-TaHRF1 vector. A schematic diagram of the vector structure is shown below. Figure 1 As shown.

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

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

[0086] With NCBI ( https: / / www.ncbi.nlm.nih.gov / Nucleotides 15108-16919 of Sequence ID: MN266288.1 from the website were used as templates for PCR amplification using primer pairs (upstream primer: 5'-ATGTTACGTCCTGTAGAA-3', SEQ ID NO. 6; downstream primer: 5'-TCATTGTTTGCCTCCCTG-3', SEQ ID NO. 7). 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 lddH2O. The amplification conditions were: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 58℃ annealing for 15 seconds, 72℃ extension for 50 seconds, repeated 32 times; 72℃ extension for 5 minutes; and holding at 16℃.

[0087] Reference - The Blunt3 Cloning Kit (Catalog No.: CB301-01, Beijing TransGen Biotech Co., Ltd.) operation steps are as follows: ligate the PCR amplification product into the cloning vector Blunt3 to obtain the pEASY-B3-GUS vector, and then perform sequencing.

[0088] Sequencing analysis revealed that the gene represented by the PCR product is the GUS gene.

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

[0090] Using pEASY-Blunt3-GUS as a template, primer pairs (upstream primer: 5'-CACCATGTTACGTCCTGTAGAA-3', SEQ ID NO.8; downstream primer: 5'-TCATTGTTTGCCTCCCTG-3', SEQ ID NO.7) were designed for PCR amplification. The amplification system consisted of 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 ddH2O to bring the total volume to 25 μl. The amplification conditions were: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 58℃ annealing for 15 seconds, 72℃ extension for 50 seconds, for 32 cycles; and 72℃ extension for 5 minutes.

[0091] The amplified PCR product was then analyzed according to pENTR. TM Directional Cloning Kit (Item No.: K2400-20SP, Thermo Scientific) TM The procedure involves ligation and sequencing. Successfully sequenced single clones are ligated to the pc186 expression vector via the LR reaction to obtain the pc186-GUS vector. A schematic diagram of the vector structure is shown below. Figure 2 As shown.

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

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

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

[0095] 1. Three days before infection, Agrobacterium pc186-TaHRF1 / EHA105 and Agrobacterium pc186-GUS / EHA105 were inoculated onto YEP solid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, respectively, and incubated in the dark at 28°C for 2 days. Single colonies were picked and inoculated into YEP liquid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, and incubated overnight at 28°C with shaking at 220 rpm. The Agrobacterium pc186-TaHRF1 / EHA105 and Agrobacterium pc186-GUS / EHA105 were transferred to sterile 2 ml centrifuge tubes, centrifuged at 6000 rpm for 5 minutes, the supernatant was discarded, and the precipitate was resuspended with resuspension buffer to obtain Agrobacterium pc186-TaHRF1 / EHA105 and Agrobacterium pc186-GUS / EHA105, respectively.

[0096] 2. Collect immature embryos (scutellum length about 2 mm) of wheat about 2 weeks after flowering from different genotypes, and infect them with Agrobacterium resuspension of pc186-TaHRF1 / EHA105 and pc186-GUS / EHA105 respectively. The embryos were laid flat on WLS-AS medium with the scutellum side facing up and incubated in the dark at 23°C for 2 days.

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

[0098] 4. Transfer the recovered callus to WLS-P5 medium and incubate in the dark at 25°C for 2 weeks.

[0099] 5. Then transfer the callus tissue to WLS-P10 medium and incubate in the dark at 25°C for 3 weeks.

[0100] 6. Transfer the above callus tissue to LSZ-P5 medium and incubate at 25°C under light for 2 weeks. Figure 3 and Figure 4 ).

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

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

[0103] Table 1: Culture medium composition for wheat genetic transformation

[0104]

[0105] II. PCR detection of candidate transgenic plants

[0106] Genomic DNA was extracted from wheat plants in the T0 generation that were transfected with the pc186-TaHRF1 and pc186-GUS vectors using the CTAB method (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 2001).

[0107] Using extracted genomic DNA from candidate transgenic plants as templates, 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 was present, the amplified fragment length was 446 bp. The total volume of the amplification system was 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. The amplification conditions were as follows: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 58℃ annealing for 15 seconds, 72℃ extension for 15 seconds, and 32 cycles of denaturation, annealing, and extension; 72℃ extension for 5 minutes.

[0108] PCR identification results as follows Figure 5 As shown, wild-type wheat does not contain a 446bp bar gene segment.

[0109] III. Statistical analysis of conversion efficiency of different wheat genotypes

[0110] Wheat embryos 14-15 days after pollination were infected with Agrobacterium, and the induced callus tissue was screened. The number of induced callus tissues was counted after transfer to LSF-P5 medium. After PCR identification, the number of transgenic positive seedlings was counted. Finally, the callus induction rate and transformation efficiency were calculated using the following formulas:

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

[0112] Conversion efficiency (%) = (Number of positive seedlings ÷ Total number of embryos) × 100%;

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

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

[0115]

[0116] Using the immature embryos of the wheat variety Liangxing 66 as explants, Agrobacterium-mediated transformation of the pc186-TaHRF1 vector and the control vector pc186-GUS resulted in callus induction rates of 95.27% and 89.63%, respectively. The transformation efficiency of the pc186-TaHRF1 vector was 45.56%, while the control vector failed to produce transgenic plants. When using the wheat cultivar Aifeng 3 as the recipient for genetic transformation, the control vector pc186-GUS failed to produce positive transgenic plants, while the transformation efficiency of the pc186-TaHRF1 vector was 39.84%. These results indicate that the TaHRF1 gene can significantly improve wheat transformation efficiency and can address some of the genotype-dependent issues in wheat genetic transformation.

[0117] IV. Phenotypic Examination of Transgenic Plants:

[0118] The growth and development of positive plants obtained from Fielder transformed with the plant expression vector pc186-TaHRF1 and wild-type Fielder plants were investigated. Results are as follows: Figure 6 As shown, the results indicate that plants overexpressing the TaHRF1 gene have normal growth and development and do not affect the agronomic traits of wheat.

[0119] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for improving the efficiency of wheat genetic transformation, characterized in that, Will TaHRF1 Genes and target nucleic acid molecules are transferred into wheat; The TaHRF1 The gene is a nucleic acid molecule as shown in SEQ ID NO.1, or a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2; The TaHRF1 Genes and target nucleic acid molecules were transferred into the wheat using the pc186 expression vector; The wheat variety in question is either Liangxing 66 or Aifeng 3.

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