TaWIP2 protein and encoding gene and application of the TaWIP2 protein and encoding gene in improving wheat genetic transformation efficiency and yield

Abstract

This invention discloses the application of TaWIP2 protein and its encoding gene in improving the genetic transformation efficiency and yield of wheat, belonging to the field of plant genetic engineering technology. The amino acid sequence of the TaWIP2 protein is shown in SEQ ID NO.2, and the nucleotide sequence of the TaWIP2 protein encoding gene is shown in SEQ ID NO.1. This invention discovers a novel TaWIP2 protein and its encoding gene involved in regulating wheat regeneration in the wheat genome, and overexpression of this protein... TaWIP2 While improving the efficiency of wheat genetic transformation, genes can also increase wheat yield, providing new genetic resources for breaking through the bottleneck of wheat genetic transformation, accelerating research on wheat gene function, and improving crop agronomic traits.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the application of a TaWIP2 protein and its encoding gene in improving the efficiency and yield of wheat genetic transformation. Background Technology

[0002] Wheat is one of the most important food crops, and genetic transformation and gene editing technologies are crucial tools for wheat functional gene research and molecular breeding. However, wheat is a crop that is relatively difficult to genetically transform. Its low regeneration efficiency, strong genotype dependence, and narrow applicability of transformation systems have long limited the improvement of the efficiency of wheat transgenic, gene editing, and molecular breeding. In particular, in common wheat, the embryonic development ability of many superior varieties or local varieties, callus tissue, or other explants is weak during in vitro culture, resulting in a low rate of positive transformation seedlings and making it difficult to meet the needs of high-throughput functional verification and precision breeding.

[0003] Existing technologies mainly utilize regeneration-related genes to improve the genetic transformation efficiency of wheat. Currently reported wheat regeneration-related genes mainly include... TaWOX5 and TaLAX1 While these genes can improve the efficiency of callus embryogenicization, bud regeneration, genetic transformation, or the acquisition of gene-edited plants, they often simultaneously participate in key growth and development processes such as cell division, meristematic maintenance, organogenesis, ear morphogenesis, fertility formation, and grain development. Therefore, if these factors are continuously expressed, excessively expressed, or expressed spatiotemporally uncontrolled in transformed materials or regenerated plants, they can easily cause problems such as plant malformation, reduced spikelet number, altered ear morphology, or fluctuations in yield-related traits.

[0004] Therefore, there is an urgent need to develop new regulatory genes suitable for wheat genetic transformation or regeneration systems, so that they can improve the efficiency of wheat genetic transformation while minimizing the adverse effects on wheat yield-related traits, thereby providing a more stable, efficient, and less agronomically trait-disrupting genetic transformation tool for wheat functional gene research and molecular design breeding. Summary of the Invention

[0005] To address the aforementioned limitations of existing technologies, the present invention aims to provide an application of the TaWIP2 protein and its encoding gene in improving wheat genetic transformation efficiency and yield. This invention identifies a novel TaWIP2 protein and its encoding gene in the wheat genome that participate in regulating wheat regeneration, and overexpresses it... TaWIP2 While improving the efficiency of wheat genetic transformation, genes can also increase wheat yield, providing new genetic resources for breaking through the bottleneck of wheat genetic transformation, accelerating research on wheat gene function, and improving crop agronomic traits.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides the application of TaWIP2 protein in improving the genetic transformation efficiency and yield of wheat; said TaWIP2 protein is a protein as shown in (A1) or (A2) below: (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 of the sequence listing; (A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0007] In the aforementioned proteins, a protein tag refers to a polypeptide or protein expressed by fusing it with the target protein using in vitro DNA recombination technology, to facilitate 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 RRRRRRR), Poly-His (typically 6 HHHHHH), FLAG (DYKDDDDK), Strep-tagII (WSHPQFEK), or c-Myc (EQKLISEEDL).

[0008] In the above applications, the TaWIP2 protein increases wheat yield by improving grain length, grain width, and thousand-grain weight.

[0009] This invention has discovered a novel wheat regeneration regulatory protein—TaWIP2—in the wheat genome. Unlike most existing wheat regeneration proteins, overexpression of TaWIP2 in wheat can simultaneously improve wheat genetic transformation efficiency and yield. Therefore, the TaWIP2 protein of this invention has important practical application value.

[0010] A second aspect of the present invention provides the application of the gene encoding the TaWIP2 protein in improving the genetic transformation efficiency and yield of wheat; said gene is TaWIP2 A gene is a DNA molecule as shown in i) or ii) below: i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) DNA molecules other than i) encoding the amino acid sequence shown in SEQ ID NO.2.

[0011] In the above applications, by promoting TaWIP2 Gene expression can be used to improve the efficiency of wheat genetic transformation and yield.

[0012] Preferred, promoting TaWIP2 The substance expressed by the gene is any one of the following: C1) contains TaWIP2 Gene expression cassettes; C2) contains TaWIP2 Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains TaWIP2 Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2); C4) contains TaWIP2 Transgenic plant cell lines containing the gene, or transgenic plant cell lines containing the expression cassette described in C1); C5) contains TaWIP2 Transgenic plant tissue containing the gene, or transgenic plant tissue containing the expression cassette described in C1); C6) contains TaWIP2 Transgenic plant organs containing genes, or transgenic plant organs containing the expression cassette described in C1).

[0013] In this invention, conventional plant expression vectors in the art can be used to construct expression vectors containing... TaWIP2 Recombinant gene expression vectors. The plant expression vectors are not particularly limited, as long as they can achieve the desired result. TaWIP2 The transcription and / or expression of genes in plant cells or plant tissues are acceptable, including but not limited to binary Agrobacterium expression vectors, plasmid vectors suitable for plant genetic transformation, and expression vectors that can be used for plant microbombardment transformation.

[0014] In some implementations, containing TaWIP2 The gene recombination vector is constructed using the following method: Will TaWIP2 Genes were inserted into the pUbi110 vector Sma I and Spe Between I sites, a structure containing TaWIP2 Gene recombination vectors.

[0015] In some embodiments, the recombinant expression vector includes [missing information - likely a specific component or vector]. TaWIP2 Gene-operable linker promoters, TaWIP2 The gene coding sequence and transcription termination sequence are specified. The promoter can be selected from constitutive promoters, inducible promoters, tissue-specific promoters, or enhancing promoters. Preferably, the promoter includes, but is not limited to, the cauliflower mosaic virus CaMV 35S promoter, the maize ubiquitin promoter, or a promoter with equivalent function. These promoters can be used alone or in combination with other plant promoters, enhancers, or regulatory elements to regulate... TaWIP2 Gene expression in plants.

[0016] In some embodiments, the plant expression vector may further include an untranslated region located at the 3' end of the exogenous gene, wherein the 3' untranslated region includes a polyadenylation signal and / or a DNA fragment involved in mRNA processing, stability regulation, or gene expression regulation. The polyadenylation signal can guide the addition of a polyadenylated tail to the 3' end of the mRNA precursor, including but not limited to the Nos terminator of the carmine synthase gene derived from the Agrobacterium Ti plasmid, the 3' untranslated region of a plant gene, or transcription termination and polyadenylation sequences with the same or similar functions.

[0017] The recombinant vector constructed in this way can be used to... TaWIP2 Genes are introduced into plant cells, tissues, callus, immature embryos, mature embryos, immature embryos, seedlings, or plants to obtain... TaWIP2 Transgenic plant materials with gene overexpression, heterologous expression, or functional verification.

[0018] In the above applications, wheat yield is increased by improving the grain length, grain width, and thousand-grain weight.

[0019] This invention has found that: overexpression TaWIP2 Genes can improve the efficiency of genetic transformation in wheat by introducing nucleic acid molecules; at the same time, they can also improve the production traits of wheat grains, such as grain length, grain width, and thousand-grain weight, thereby increasing wheat yield.

[0020] The beneficial effects of this invention are: The present invention TaWIP2 Genes play a significant promoting role in the genetic transformation of wheat. By... TaWIP2 The CDS sequence of a gene was constructed into a plant overexpression vector and introduced into wheat embryos via Agrobacterium-mediated transformation. This facilitates the entry of exogenous nucleic acid molecules into wheat recipient cells, increasing the efficiency of positive transformation events and thus improving the efficiency of wheat genetic transformation. This technology provides a new functional gene and technical means to address the problems of low genetic transformation efficiency in monocotyledonous plants such as wheat and the significant genotypic limitations of transformation systems.

[0021] More importantly, the present invention TaWIP2 While improving the efficiency of wheat genetic transformation, the gene did not show any adverse effects on wheat yield traits; on the contrary, it improved grain-related yield traits in transgenic wheat. Compared with the control vector, TaWIP2 The wheat grains of the gene-overexpressing material showed increased grain length, grain width, and thousand-grain weight, indicating that... TaWIP2 Genes can not only serve as functional factors to improve the efficiency of wheat genetic transformation, but also as candidate genes to improve wheat grain traits and increase yield potential.

[0022] Therefore, the present invention TaWIP2Genes have both transformation-promoting and yield-improving functions, and are of great application value in wheat gene function research, genetic transformation system optimization, molecular breeding, and the creation of high-yield wheat germplasm. Attached Figure Description

[0023] Figure 1 This is a partial structural diagram of the plant expression vector pUbi110-TaWIP2.

[0024] Figure 2 This is a partial structural diagram of the plant expression vector pUbi110-GUS.

[0025] Figure 3 PCR-specific amplification of candidate transgenic plants obtained by transfecting wheat with the plant expression vector pUbi110-TaWIP2 bar Gene results; Figure 1-13 represents candidate transgenic plants, PC represents positive plasmid, WT represents wild-type control, NC represents negative control, and M represents 2000 bp molecular weight marker.

[0026] Figure 4 Statistical results of genetic transformation efficiency of plant expression vectors pUbi110-TaWIP2 and pUbi110-GUS infecting Sumai 3.

[0027] Figure 5 This figure shows the statistical results of seed length in Fielder plants transformed with the plant expression vector pUbi110-TaWIP2. WT represents the wild-type control, and OE#2 and OE#8 represent transgenic plants. represent p <0.05.

[0028] Figure 6 This figure shows the statistical results of grain width in Fielder plants transformed with the plant expression vector pUbi110-TaWIP2. WT represents the wild-type control, and OE#2 and OE#8 represent transgenic plants. represent p <0.05.

[0029] Figure 7 The figure shows the statistical results of the thousand-seed weight of Fielder plants transformed with the plant expression vector pUbi110-TaWIP2. WT represents the wild-type control, and OE#2 and OE#8 represent transgenic plants. represent p <0.05. Detailed Implementation

[0030] It should be noted that the following detailed descriptions are illustrative 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.

[0031] As mentioned earlier, wheat is a crop that is relatively difficult to genetically transform. Its low regeneration efficiency, strong genotype dependence, and narrow applicability of transformation systems have long limited the improvement of wheat transgenic, gene editing, and molecular breeding efficiency. Although existing wheat regeneration genes can improve the efficiency of callus embryogenicization, bud regeneration, genetic transformation, or gene-edited plant acquisition, they may have adverse effects on wheat yield traits.

[0032] WIP proteins are a class of wound-inducing proteins. Current research on WIP family genes mainly focuses on model plants such as Arabidopsis thaliana, and their biological functions in wheat, an important food crop, have not yet been reported.

[0033] in wheat TaWIP2 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the amino acid sequence of the TaWIP2 protein is shown in SEQ ID NO.2, as follows: TaWIP2 The nucleotide sequence of the gene:

[0034] The amino acid sequence of the TaWIP2 protein: MEDPYTSFLKNPYYYYCTSSFPTAPPTPHLPPPFQPYAALYPAVAAAAAPHHQYPSFFQHQPAQPTHHYSTAPPSPPLREALPLLSLSPTPAARPRAVQHHDAADSDSDDDNNDCCYHLRQEVAAGSRTTSARTPLFADLNCVPSCCDDGDGDPMDVEASWSTSTDDAAVALRIGLPAAEADLLSGLSGRAAEEEEDDCGGGH EEVPLGFSTAPIGRLNKGQYWIPTPSQILIGPTQFSCPVCFKTFNRYNNMQMHMWGHGSQYRKGPESLRGVQPTAMLRLPCYCCAAGCRNNIDHPRAKPLKDF RTLQTHYKRKHGLKPFLCRRCGKAFAVKGDWRTHEKNCGKLWYCLCGSEFKHKRSLKDHARAFGHGHGAFGCNGAAGGDGSFDDDDEGAVSEIEHDVVCGAAAR.

[0035] To investigate the function of the TaWIP2 protein in wheat, this invention constructed a recombinant expression vector containing the TaWIP2 protein-coding gene. This recombinant expression vector was transformed into *Agrobacterium tumefaciens*, and then used to infect wheat immature embryo explants. Results showed that overexpression... TaWIP2 Genes can improve the efficiency of genetic transformation in wheat embryos; they can also improve wheat yield traits. Therefore, TaWIP2 This invention is proposed because it can serve as a new wheat regeneration gene with great value for production applications.

[0036] 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.

[0037] The test materials used in the embodiments of this invention, unless otherwise specified, are all conventional test materials in the art and can be purchased through commercial channels. Where specific experimental conditions and methods are not specified in the embodiments of this invention, conventional conditions are generally followed.

[0038] Example 1: Containing TaWIP2 Construction of gene recombination vectors Total RNA was extracted from the Fielder wheat variety using the Ultrapure RNA Kit (Kangwei Century, catalog number: CW0581S). cDNA was then reverse transcribed using the HiScript® II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Nanjing Novizan Biotechnology Co., Ltd., catalog number: R223-01).

[0039] Using cDNA as a template, in TaWIP2 Primer pairs (upstream primer: 5'-GTAGCCAAGAAGAGAGAAGC-3', SEQ ID NO.3; downstream primer: 5'-TAGTGTTTGCTCGTGCGTG-3', SEQ ID NO.4) were designed for PCR amplification of the 5'-UTR and 3'-UTR regions of the gene. 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, 60℃ annealing for 15 seconds, 72℃ extension for 40 seconds, for 40 cycles; and 72℃ extension for 5 minutes.

[0040] The amplified PCR product was ligated into the pEASY®-Blunt3 vector according to the operating steps of the pEASY®-Blunt3 Cloning Kit (Beijing TransGen Biotech Co., Ltd., catalog number: CB301-02). After sequencing verification, the pEASY-Blunt3-TaWIP2 vector was obtained.

[0041] Select a single restriction endonuclease cleavage site on the overexpression vector pUbi110 ( Sma I Spe I) Perform vector digestion to obtain the digestion product. Then, perform gel recovery using the gel recovery / PCR product purification kit (Shandong Cisco Biotechnology Co., Ltd., catalog number: AE0101-C).

[0042] Using pEASY-Blunt3-TaWIP2 plasmid as a template, homologous primer pairs (upstream primer: 5'-CAGGTCGACTCTAGAGGATCCATGGAAGACCCCTACACGAGC-3', SEQ ID NO.5; downstream primer: 5'-GAATTCCGGCTCGAGACTAGTCCGCGCGGCGGCGCCGCA-3', SEQ ID NO.6) 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 to bring the total volume to 25 μl. The amplification conditions were: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 60℃ annealing for 15 seconds, 72℃ extension for 45 seconds, for 40 cycles; and 72℃ extension for 5 minutes.

[0043] The amplified PCR product was ligated to the pUbi110 vector digestion product using the homologous recombination steps of 2x Ezmax® Ultra Universal Clone Mix (Shanghai Tulugang Biotechnology Co., Ltd.) and sequenced. Sequencing analysis showed that the nucleotide sequence of the PCR amplified product was sequence 1 in the sequence listing, and the amino acid sequence of the protein encoded by this gene was sequence 2 in the sequence listing. Plasmids were extracted from the correctly sequenced single clones using the SPARKeasy Plasmid Mini-Range Rapid Extraction Kit (Shandong Cisco Biotechnology Co., Ltd., catalog number: AD0101-C) to construct the gene containing... TaWIP2 The recombinant vector (pUbi110-TaWIP2) is shown in the schematic diagram of part of the vector structure. Figure 1 As shown.

[0044] Example 2: Construction of control vector pUbi110-GUS Nucleotides 15108-16919 of Sequence ID MN266288.1 on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) were used for PCR amplification using primer pairs (upstream primer: 5'-ATGTTACGTCCTGTAGAA-3', SEQ ID NO.7; downstream primer: 5'-TCATTGTTTGCCTCCCTG-3', SEQ ID NO.8). 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 5 minutes; 95℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 1 minute, for 38 cycles; and 72℃ extension for 5 minutes.

[0045] The amplified PCR product was ligated according to the operating steps of the pEASY®-Blunt3 Cloning Kit (catalog number: CB301-02, Beijing TransGen Biotech Co., Ltd.), and the result was verified by sequencing to obtain pEASY-Blunt3-GUS.

[0046] Using pEASY-Blunt3-GUS as a template, primer pairs (upstream primer: 5'-CGACTCTAGAGGATCCCCGGGATGTTACGTCCTGTAGAAACCCCA-3', SEQ ID NO.9; downstream primer: 5'-GAATTCCGGCTCGAGACTAGTTTGTTTGCCTCCCTGCTGC-3', SEQ ID NO.10) 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 was added to bring the total volume to 25 μl. The amplification conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for 38 cycles; and 72℃ extension for 5 min.

[0047] The amplified PCR product was ligated into the pUbi110 vector digestion product according to the homologous recombination steps of 2x Ezmax® Ultra Universal Clone Mix (Shanghai Tulugang Biotechnology Co., Ltd.) and sequenced. Single clones with correct sequencing were used to extract plasmids using the SPARKeasy plasmid mini-scale rapid extraction kit (Shandong Cisco Biotechnology Co., Ltd., catalog number: AD0101-C) to obtain the pUbi110-GUS vector. A partial structural diagram of the vector is shown below. Figure 2 As shown.

[0048] Example 3: Agrobacterium-mediated genetic transformation of wheat and evaluation of transformation efficiency 1. Test method: For detailed steps and methods of Agrobacterium-mediated genetic transformation of wheat, please refer to Wheat ( Triticum aestivum L.) Transformation Using Immature Embryos (Ishida et al., 2015). The basic steps of genetic transformation are as follows: (1) The mixture containing the material constructed in Example 1 TaWIP2 The recombinant vector (pUbi110-TaWIP2) was transformed into Agrobacterium EHA105 competent cells to obtain a recombinant Agrobacterium strain suitable for transformation, named pUbi110-TaWIP2 / EHA105; the control vector pUbi110-GUS constructed in Example 2 was transformed into Agrobacterium EHA105 competent cells to obtain a recombinant Agrobacterium strain suitable for transformation, named pUbi110-GUS / EHA105.

[0049] (2) Three days before infection, Agrobacterium tumefaciens pUbi110-TaWIP2 / EHA105 and pUbi110-GUS / EHA105 were inoculated onto YEP solid medium containing 50 mg / L kanamycin and 50 mg / L rifampin, 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 rifampin, and cultured overnight at 220 rpm with shaking at 28°C. The above Agrobacterium tumefaciens solution was transferred into 2 ml centrifuge tubes, centrifuged at 6000 rpm for 5 minutes, the supernatant was discarded, and the precipitate was resuspended with resuspension to obtain Agrobacterium tumefaciens resuspensions of pUbi110-TaWIP2 / EHA105 and pUbi110-GUS / EHA105, respectively.

[0050] (3) The embryos of “Sumai No. 3” wheat 14 days after flowering were infected with Agrobacterium resuspension of pUbi110-TaWIP2 / EHA105 and pUbi110-GUS / EHA105 respectively. The embryos were spread flat on WLS-AS medium (1 / 10 MS basic medium, 1 / 10 MS vitamins, glucose 10 g / L, acetylsuccinone 100 μM, agarose 8 g / L) with the scutellaria side up, and cultured for 2 days in the dark at 23°C.

[0051] (4) After co-culture, the embryos were transferred to WLS-Res medium (MS basal 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 the dark at 25°C for 5 days.

[0052] (5) Transfer the recovered callus to WLS-P5 medium (WLS-Res medium with 5 mg / L PPT (glufosinate)) and culture in the dark at 25°C for 14 days.

[0053] (6) The callus tissue was then transferred to WLS-P10 medium (WLS-Res medium with 10 mg / L PPT added) and cultured in the dark at 25°C for 21 days.

[0054] (7) Transfer the above callus tissue to LSZ-P5 medium (MS basal medium, LS vitamins, zeatin 5 mg / L, sucrose 20 g / L, carbenicillin 250 mg / L, PPT 5 mg / L, plant gel 3 g / L) and culture in a 25°C incubator under light for 2 weeks.

[0055] (8) The regenerated shoots of wheat callus were transferred to LSF-P5 medium (MS basal medium, LS vitamins, IBA 0.2 mg / L, sucrose 15 g / L, carbenicillin 250 mg / L, PPT 5 mg / L, plant gel 3 g / L) and cultured in a 25°C incubator under light until the roots of the regenerated shoots were about 1-2 cm long. Then, the strong seedlings with long roots were transplanted into nutrient soil to obtain candidate transgenic plants.

[0056] The candidate transgenic plants were identified by PCR using the following method: Genomic DNA was extracted from leaves of wheat plants transformed with pUbi110-TaWIP2 and pUbi110-GUS vectors in generation T0 using the CTAB method. Primers were designed for detection. bar Gene and primer pair sequences (upstream primer: 5'-GGCGGTCTGCACCATCGTCAACCACTAC-3', SEQ ID NO.11; downstream primer: 5'-AGTCCAGCTGCCAGAAACCCACGTCATG-3', SEQ ID NO.12). The amplification system consisted of 1 μl upstream primer (10 μmol / μl), 1 μl downstream primer (10 μmol / μl), 10 μl 2×Rapid Taq Master Mix, 1 μl cDNA template, and ddH2O to bring the total volume to 20 μl. Amplification conditions were: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 10 seconds, for 35 cycles; 72℃ extension for 5 minutes. After PCR, gel electrophoresis was performed using 1% agarose gel.

[0057] After wheat embryos are infected with Agrobacterium, the induced callus tissue is screened and differentiated, and the resulting resistant shoots are transferred to a seedling culture medium. Candidate transgenic plants are identified by PCR, the number of positive seedlings is counted, and the transformation efficiency is calculated.

[0058] Conversion efficiency (%) = Number of positive seedlings ÷ Total number of embryos × 100%.

[0059] 2. Test Results: The results showed that, with the wild type as a blank control, a total of 10 positive plants were found among some candidate transgenic plants. Figure 3 ).

[0060] The statistical results of conversion efficiency are as follows: Figure 4 As shown, the transformation efficiency of the control vector was 4.20%, while the transformation efficiency of the pUbi110-TaWIP2 vector was 61.27%, which was significantly higher than that of the control.

[0061] The above results indicate that overexpression TaWIP2 Genes can significantly improve the efficiency of genetic transformation in wheat.

[0062] Example 4: Overexpression TaWIP2 Effects of genes on wheat yield traits 1. Test method: Using Fielder as background material, and referring to the Agrobacterium-mediated method in Example 3, wheat containing... TaWIP2The recombinant vector (pUbi110-TaWIP2) was transformed into Fielder cells to obtain transgenic plants. After positive PCR identification, the grain length, grain width, and thousand-grain weight of the self-pollinated homozygous transgenic plants were statistically analyzed.

[0063] 2. Test Results: The results showed that the average seed length of transgenic plant OE#2 was 6.37 mm, that of transgenic plant OE#8 was 6.36 mm, and that of wild-type plant was 6.15 mm; the increase in seed length was significantly different between the two transgenic plants and the wild-type plant. Figure 5 The average seed width of transgenic plant OE#2 was 3.50 mm, and that of OE#8 was 3.57 mm, while the average seed width of wild-type plants was 3.19 mm. Both transgenic plants showed significant differences in seed width compared to the wild type. Figure 6 The average thousand-grain weight of transgenic plants OE#2 was 49.08 g, and that of OE#8 was 51.84 g, while the average thousand-grain weight of wild-type plants was 47.61 g. Both transgenic plants showed significant differences in thousand-grain weight compared to the wild type. Figure 7 The above results indicate that... TaWIP2 Genes can improve the length, width, and thousand-grain weight of wheat grains, which has important economic value and social benefits for improving crop agronomic traits and modern agricultural production.

[0064] 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. The application of TaWIP2 protein in improving wheat genetic transformation efficiency and yield, characterized in that, The TaWIP2 protein is the protein shown in either (A1) or (A2) below: (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 of the sequence listing; (A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

2. The application according to claim 1, characterized in that, The TaWIP2 protein increases wheat yield by improving grain length, grain width, and thousand-grain weight.

3. The application of the gene encoding the TaWIP2 protein in improving wheat genetic transformation efficiency and yield, characterized in that... The encoding gene is TaWIP2 A gene is a DNA molecule as shown in i) or ii) below: i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) DNA molecules other than i) encoding the amino acid sequence shown in SEQ ID NO.

2.

4. The application according to claim 3, characterized in that, By promoting TaWIP2 Gene expression can be used to improve the efficiency of wheat genetic transformation and yield.

5. The application according to claim 4, characterized in that, Promote TaWIP2 The substance expressed by the gene is any one of the following: C1) contains TaWIP2 Gene expression cassettes; C2) contains TaWIP2 Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains TaWIP2 Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2); C4) contains TaWIP2 Transgenic plant cell lines containing the gene, or transgenic plant cell lines containing the expression cassette described in C1); C5) contains TaWIP2 Transgenic plant tissue containing the gene, or transgenic plant tissue containing the expression cassette described in C1); C6) contains TaWIP2 Transgenic plant organs containing genes, or transgenic plant organs containing the expression cassette described in C1).

6. The application according to claim 5, characterized in that, contain TaWIP2 The gene recombination vector is constructed using the following method: Will TaWIP2 Genes were inserted into the pUbi110 vector Sma I and Spe Between I sites, a structure containing TaWIP2 Gene recombination vectors.

7. The application according to claim 4, characterized in that, Wheat yield can be increased by improving the grain length, grain width, and thousand-grain weight.

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