Application of TaAGD gene in promotion of wheat regeneration and genetic transformation

Abstract

The invention discloses application of a TaAGD gene in promoting wheat regeneration and genetic transformation, and belongs to the technical field of plant genetic engineering. According to the present invention, the first research discovers that the wheat regeneration efficiency can be improved by overexpressing the TaAGD gene in the wheat immature embryo explant, such that the wheat genetic transformation process is promoted, the new regeneration gene resource is provided for the wheat biological breeding, and the important significance is provided for the promotion of the wheat genetic improvement.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to an application of a TaAGD gene in promoting wheat regeneration and genetic transformation. Background Art

[0002] Wheat (Triticum aestivum L.) is a major staple crop in my country, accounting for 40% of total staple food. High and stable wheat yields are essential for ensuring national food security. Biotechnology-based breeding has gradually become an important development direction for future wheat breeding, and genetic transformation and gene editing are important methods for wheat gene discovery, functional research, and mechanistic analysis. Genetic transformation primarily relies on wheat's ability to regenerate in vitro. Wheat is an allohexaploid plant with a large genome and numerous repetitive sequences, making genetic transformation difficult and a major limitation to the slow progress of wheat biobreeding.

[0003] In recent years, wheat genetic transformation technology has made significant progress. Studies have shown that overexpressing key genes that promote regeneration can effectively improve the regeneration ability and genetic transformation efficiency of crops. This method has gradually become a new strategy to improve the efficiency of wheat genetic transformation. However, the number of regeneration-related genes isolated and identified is still limited, which to some extent restricts the further application of this technology. Therefore, in-depth exploration and identification of more key genes that promote wheat regeneration will not only help improve the agronomic traits of crops, but will also provide important theoretical support and genetic resources for biotechnology breeding, which is of great significance to promoting wheat genetic improvement.

[0004] Arf-GAP (ADP-ribosylation factor GTPase-activating protein) is a class of proteins containing the Arf-GAP domain, whose main function is to regulate the activity of the small G protein Arf (ADP-ribosylation factor). Arf proteins play an important role in intracellular vesicle transport, membrane dynamics and signal transduction, and Arf-GAP regulates multiple biological processes by promoting the conversion of Arf proteins from the GTP-bound state (active state) to the GDP-bound state (inactive state). Studies have found that Arabidopsis AtAGD1 (Arf-GAP domain-containing protein 1) plays an important role in intracellular membrane transport and cell wall synthesis. AtAGD4 is involved in the regulation of cell wall synthesis and cell morphology (Sun Jinhao, Chinese Academy of Agricultural Sciences, 2021). Other AGD genes are involved in many aspects of plant stress response. However, there is currently relatively little research on the function of Arf-GAP in wheat, and its function in promoting wheat regeneration and genetic transformation has not yet been reported. Summary of the Invention

[0005] In response to the above-mentioned prior art, the present invention aims to provide a method for using the TaAGD gene to promote wheat regeneration and genetic transformation. The present invention discovers a new TaAGD gene in the wheat genome that is involved in regulating the wheat regeneration process. Overexpression of the TaAGD gene can improve the regeneration efficiency of wheat immature embryo explants and promote the genetic transformation process of wheat.

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

[0007] In a first aspect of the present invention, a use of the TaAGD gene in improving the regeneration efficiency of wheat immature embryo explants is provided; the TaAGD gene is a DNA molecule as shown in the following i) or ii):

[0008] i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1;

[0009] ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO. 2 except i).

[0010] The present invention found that overexpression of the TaAGD gene can increase the number of wheat immature embryo explants that form resistant callus and differentiate into positive seedlings, and can be used as a new wheat regeneration gene to improve the regeneration efficiency of wheat immature embryos.

[0011] The second aspect of the present invention provides the use of TaAGD protein in improving the regeneration efficiency of wheat immature embryos.

[0012] In the above application, the TaAGD protein is the protein shown below (A1) or (A2):

[0013] (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO. 2 in the sequence listing;

[0014] (A2) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0015] The third aspect of the present invention provides the use of a recombinant expression vector or genetically engineered bacteria containing the TaAGD gene in improving the regeneration efficiency of wheat immature embryo explants.

[0016] A fourth aspect of the present invention provides a method for improving the regeneration efficiency of wheat immature embryo explants, comprising the following steps:

[0017] The TaAGD gene is connected to an expression vector to construct a recombinant expression vector, which is then transferred into Agrobacterium competent cells to obtain an Agrobacterium strain for transformation; and the Agrobacterium strain is used to infect wheat immature embryos.

[0018] Preferably, the expression vector is a pUbi110 vector; the TaAGD gene is linked between the SmaI and SpeI sites of the pUbi110 vector.

[0019] Preferably, the Agrobacterium competent cells are Agrobacterium tumefaciens EHA105.

[0020] Beneficial effects of the present invention:

[0021] The present invention is the first to discover that overexpressing the TaAGD gene in wheat immature embryo explants can improve the regeneration efficiency of wheat immature embryos, thereby promoting the genetic transformation process of wheat, providing a new regeneration gene resource for wheat genetic breeding, and is of great significance for promoting the genetic improvement of wheat. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the plant expression vector Ubi-TaAGD vector structure.

[0023] Figure 2 Schematic diagram of the plant expression vector Ubi-GUS vector structure.

[0024] Figure 3 Schematic diagram of PCR-specific amplification of the bar gene in candidate transgenic plants transformed with the plant expression vector Ubi-TaAGD. Figures 1-9 represent candidate transgenic plants transformed with Shi 4185. PC represents the positive plasmid, NC represents the negative control, WT represents the wild-type control, and M represents a 2000 bp molecular weight marker.

[0025] Figure 4 The callus differentiation rate of immature embryos of wheat variety Shi 4185 infected with plant expression vector Ubi-TaAGD and control vector Ubi-GUS.

[0026] Figure 5 The genetic transformation efficiency of the plant expression vector Ubi-TaAGD and the control vector Ubi-GUS in infecting the immature embryos of the wheat variety Shi 4185. DETAILED DESCRIPTION

[0027] 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.

[0028] As mentioned above, genetic transformation of wheat primarily relies on its ability to regenerate in vitro. Wheat is an allohexaploid plant with a large genome and numerous repetitive sequences, making genetic transformation difficult and a major limitation to the slow progress of wheat biobreeding. Overexpressing key genes that promote regeneration can effectively enhance wheat regeneration capacity and genetic transformation efficiency, but the number of genes currently reported to promote wheat regeneration is limited, and further research and identification of key genes that can promote wheat regeneration is needed.

[0029] In light of this, the present invention conducted an in-depth study of the wheat genome and discovered a hypothetical protein of unknown function, CFC21_093461, in GenBank. This protein contains an Arf-GAP domain. The present invention named this hypothetical protein TaAGD protein and the gene encoding it TaAGD gene.

[0030] The nucleotide sequence of the TaAGD gene is shown in SEQ ID NO. 1, and is as follows:

[0031] ATGAGTGCTGCTAATCGTTCCCAACCCATCAAGTTGAACAGGCCTGTCGTAGGCAAAGCACGAAAGTTGAAGGATCTCATGCTGAAAAGTGACAATCGAGTGTGTGCTGATTGTAGTGCACCTGACCCCAAATGGGCGTCTTCTAATATCGGAGTATTTCTTTGCTTAAAATGTGGAGACGTCCACAGGGCCCTTGGACAAGACATTTCAAACGTTTTGTCTTTAACTTTGGATGATTGGTCTGATAGTGATATTGACTCCATGATTGAGGTTGGTGGAAACTCATATGCAAATTCAATTTATGAGGCTTTTCTTCCAAAAGATCACCCAAAACCTAAACCAGACTCACCAATGGAATATCGTACCAAATTTATAAGAGCCAAGTATGAAACACAAGATTTTCTGAAGCCAAGTTTGCGCATTAGCTCAAAGGCAGGTTTAGAATCTACCAATTCTCTGAACAGTGTGGATAATAGTTTCTCTAGCACTTCAAGGAAGCATGCCCCAGAAGATACAAGAGAATTTGTTGGACAACTGAACATTACAGTGGTAAAAGGTTCTGGGTTGGCGGTCAGAGATATGCTTACAAGTGATCCTTATGTTGTTTTAAGTCTTGGAGAGCAGAAGGCTCAAACAACAGTTAAAGCGAGTGACCTGAACCCGGAAGTGTATGATCACGACACTTTTTCTGCTGACGATATCATGGGGGAAGCAGAGATAGATCTGAAGCCAATGATCACAGCTGCTATGGCCTTTGGAGACCCGTCGCGTCACGCGGACATGCAAATTGGAAGGTGGTTCATGACCAGAGACAATTGCCTGTTGAGCGACAGCATTGTCAATATTTCGTCGGGAAAGGTAAAACAGGAAGTTTACCTAAAGCTGCAGAACGTAGAATCAGGTGAGATGGAGTTAGAACTGGAATGGGCTCGTCTAGATTAA。

[0032] The amino acid sequence of the TaAGD protein is shown in SEQ ID NO. 2, and is as follows:

[0033] MSAANRSQPIKLNRPVVGKARKLKDLMLKSDNRVCADCSAPDPKWASSNIGVFLCLKCGDVHRALGQDISNVLSLTLDDWSDSDIDSMIEVGGNSYANSIYEAFLPKDHPKPKPDSPMEYRTKFIRAKYETQDFLKPSLRISSKAGLESTNSLNSVD NSFSSTSRKHAPEDTREFVGQLNITVVKGSGLAVRDMLTSDPYVVLSLGEQKAQTTVKASDLNPEVYDHDTFSADDIMGEAEIDLKPMITAAMAFGDPSRHADMQIGRWFMTRDNCLLSDSIVNISSGKVKQEVYLKLQNVESGEMELELEWARLD.

[0034] To investigate the function of the TaAGD protein, the present inventors constructed a recombinant expression vector containing the TaAGD gene, transformed the recombinant expression vector into Agrobacterium tumefaciens, and then infected wheat immature embryo explants. The results showed that overexpression of the TaAGD gene enhanced the regeneration and genetic transformation efficiency of wheat immature embryos. Therefore, the TaAGD gene can be used as a novel wheat regeneration gene, which led to the present invention.

[0035] 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.

[0036] All experimental materials used in the examples of the present invention that are not specifically described are conventional experimental materials in the art and can be purchased through commercial channels. Where specific experimental conditions and methods are not specified in the examples of the present invention, conventional conditions, such as those in J. Sambrook et al., ed., Molecular Cloning Laboratory Manual (3rd Edition), Science Press, 2002; and D.L. Spector et al., ed., Cell Experiment Manual, Science Press, 2001, were generally followed, or the conditions recommended by the manufacturer.

[0037] Example 1: Construction of plant expression vectors Ubi-TaAGD and Ubi-GUS

[0038] 1. Construction of plant expression vector Ubi-TaAGD:

[0039] Total RNA was extracted from wheat callus using the Ultrapure RNA Kit (Cornwell, Catalog No. CW0581M). The extracted RNA was then reverse transcribed into cDNA using the FastKing RT Kit (with gDNase) (Tiangen Biochemical Technology (Beijing) Co., Ltd., Catalog No. KR116). Specific amplification primers were designed based on the TaAGD gene sequence from Blast, and sequence amplification was performed using a high-fidelity enzyme (Nanjing Novozymes Biotechnology Co., Ltd., Catalog No. P515). The amplification primer sequences were:

[0040] TaAGD-F: ATGAGTGCTGCTAATCGTTCC;

[0041] TaAGD-R:TTAATCTAGACGAGCCCATTCC.

[0042] Reference The amplified product was recovered using the Blunt3 Cloning Kit (Beijing Quanshijin Biotechnology Co., Ltd., Catalog No. CB301-01), ligated to the Blunt3 vector, and transformed into E. coli DH5α. Single clones were selected for sequencing. Correctly sequenced clones were then plasmid extracted using the FastPure Plasmid Mini Kit (Nanjing Novogene Biotechnology Co., Ltd., Catalog No. DC201-01) to generate the pEASY-Blunt3-TaAGD plasmid.

[0043] Using pEASY-Blunt3-TaAGD plasmid as template, a homologous recombination primer pair was designed (upstream primer: 5'-cgactctagaggatc cccggg ATGAGTGCTGCTAATCGTTCCC-3'; downstream primer: 5'-gaaatccggctcgag actagt TTAATCTAGACGAGCCCATTCCA-3') was amplified by PCR. The amplified PCR product was detected by LightNing. tM DNAAssembly Mix Plus (Jiangsu Yugong Life Science Co., Ltd.) was used for homologous recombination and ligated with the pUbi110 vector digestion product and sequenced. The correct single clone was extracted with the FastPure Plasmid Mini Kit (Nanjing Novozymes Biotech Co., Ltd., Cat. No. DC201-01) to obtain the plant expression vector Ubi-TaAGD. The schematic diagram of the vector structure is shown in the figure. Figure 1 shown.

[0044] 2. Construction of plant expression vector Ubi-GUS:

[0045] The nucleotides 15108-16919 of Sequence ID: MN266288.1 on the NCBI (https: / / www.ncbi.nlm.nih.gov / ) website were used as templates and amplified using a high-fidelity enzyme (Nanjing Novozymes Biotech Co., Ltd., Cat. No.: P515). The primer sequences were:

[0046] GUS-F:ATGTTACGTCCTGTAGAA;

[0047] GUS-R: TCATTGTTTGCCTCCCTG.

[0048] Reference The amplified product was recovered using the Blunt3 Cloning Kit (Beijing Quanshijin Biotechnology Co., Ltd., Catalog No. CB301-01), ligated to the Blunt3 vector, and transformed into E. coli DH5α. Single clones were selected for sequencing. Correctly sequenced single clones were extracted using the FastPure Plasmid Mini Kit (Nanjing Novozymes Biotechnology Co., Ltd., Catalog No. DC201-01) to generate the pEASY-Blunt3-GUS plasmid.

[0049] Using pEASY-Blunt3-GUS plasmid as template, homologous recombination primers were designed (upstream primer: 5'-cgactctagaggatc cccggg ATGTTACGTCCTGTAGAAACCCCA-3'; downstream primer: 5'-gaaatccggctcgag actagt TTGTTTGCCTCCCTGCTGC-3') was PCR amplified. The PCR product obtained by amplification was detected by referring to LightNing TM DNAAssembly Mix Plus (Jiangsu Yugong Life Science Co., Ltd.) was used for homologous recombination and ligated with the pUbi110 vector digestion product and sequenced. The correct single clone was extracted with the FastPure Plasmid Mini Kit (Nanjing Novozymes Biotech Co., Ltd., Cat. No. DC201-01) to obtain Ubi-GUS. Figure 2 shown.

[0050] Ubi-GUS was transformed into Agrobacterium EHA105 competent cells, and an Agrobacterium strain suitable for transformation was obtained, named Ubi-GUS / EHA105.

[0051] Example 2: Agrobacterium-mediated transformation of wheat immature embryos and identification of transgenic plants

[0052] 1. Agrobacterium-mediated transformation of wheat immature embryos:

[0053] The plant expression vector Ubi-TaAGD constructed in Example 1 was transformed into Agrobacterium EHA105 competent cells, and an Agrobacterium strain suitable for transformation was obtained, which was named Ubi-TaAGD / EHA105.

[0054] The plant expression vector Ubi-GUS constructed in Example 1 was transformed into Agrobacterium EHA105 competent cells, and an Agrobacterium strain suitable for transformation was obtained, which was named Ubi-GUS / EHA105.

[0055] The transformed Agrobacterium strain was used to infect wheat embryos. The detailed steps and methods of the Agrobacterium-mediated infection of wheat embryos were referenced by Ishida et al. (Ishida et al., 2015), as follows:

[0056] (1) Three days before infection, Ubi-TaAGD / EHA105 and Ubi-GUS / EHA105 Agrobacterium were inoculated onto YEP solid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, respectively, and cultured in a dark incubator at 28°C for 2 days. A single colony was picked and inoculated into YEP liquid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, and cultured overnight at 28°C and 220 rpm. The above Agrobacterium suspension was placed into a sterile 2 ml centrifuge tube, centrifuged at 6000 rpm for 5 minutes, the supernatant was discarded, and the precipitate was resuspended with resuspension buffer to obtain Ubi-TaAGD / EHA105 and Ubi-GUS / EHA105 Agrobacterium resuspensions, respectively.

[0057] (2) The immature embryos of "Shi 4185" wheat were collected about 2 weeks after flowering and infected with Agrobacterium resuspensions of Ubi-TaAGD / EHA105 and Ubi-GUS / EHA105, respectively. The embryos were spread with the scutellum side facing up on WLS-AS medium (1 / 10MS basic medium, 1 / 10MS vitamins, 10 g / L glucose, 100 μM acetosyringone, 8 g / L agarose), and cultured in a dark incubator at 23°C for 2 days.

[0058] (3) After co-culture, 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.

[0059] (4) The callus tissue after recovery culture was transferred to WLS-P5 medium (WLS-Res medium supplemented with PPT 5 mg / L) and cultured in a dark incubator at 25°C for 2 weeks.

[0060] (5) The callus tissue was then transferred to WLS-P10 medium (WLS-Res medium supplemented with 10 mg / L PPT) and cultured in a dark incubator at 25°C for 3 weeks.

[0061] (6) The callus tissue was transferred to LSZ-P5 medium (MS basic medium, LS vitamins, zeatin 5 mg / L, sucrose 20 g / L, carbenicillin 250 mg / L, PPT 5 mg / L, phytagel 3 g / L) and cultured in a 25°C incubator under light for 2 weeks.

[0062] (7) The regenerated resistant buds of wheat were transferred 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, plant gel 3 g / L) and cultured in a 25°C incubator under light until the roots of the regenerated buds were about 1-2 cm long.

[0063] (5) The strong seedlings with roots were transplanted into nutrient soil to obtain candidate transgenic seedlings of Ubi-TaAGD and Ubi-GUS, respectively.

[0064] 2. PCR detection of candidate transgenic plants:

[0065] The CTAB method (Sambrook and Russell, Molecular Cloning Laboratory Manual, 2001) was used to extract genomic DNA from T0 generation wheat plants transformed with Ubi-TaAGD / EHA105 and Ubi-GUS / EHA105 vectors.

[0066] The extracted genomic DNA of the candidate transgenic plants was used as a template for PCR amplification using Taq DNA polymerase (Nanjing Novozymes Biotech Co., Ltd., Cat. No.: P222). The primer sequences were:

[0067] bar-F: GGCGGTCTGCACCATCGTCAACCACTAC;

[0068] bar-R: AGTCCAGCTGCCAGAAACCCACGTCATG.

[0069] Detect the presence of the glyphosate resistance gene bar. If the bar gene is present, the amplified fragment length is 446 bp. Figure 3 As shown, a 446 bp-long fragment could be amplified in candidate transgenic plants 1-9, all of which were positive plants; the negative control and wild-type wheat did not have a 446 bp-long bar gene fragment.

[0070] 3. Statistics of transformation efficiency of different wheat genotypes:

[0071] After the immature embryos of wheat "Shi 4185" were infected with Agrobacterium, the calli induced and formed were screened. When they were transferred to LSF-P5 medium, the number of differentiated calli was counted. After PCR identification, the number of positive seedlings was counted, and the callus differentiation rate and transformation efficiency were finally calculated. The calculation formula is as follows:

[0072] Callus differentiation rate (%) = (number of differentiated calli ÷ total number of immature embryos) × 100%;

[0073] Transformation efficiency (%) = (number of positive seedlings ÷ total number of embryos) × 100%.

[0074] The efficiency of genetic transformation of wheat is greatly affected by the genotype, and the difficulty of genetic transformation of different genotypes of wheat varies. The wheat variety "Shi 4185" is one of the wheat genotypes that is more difficult to transform. Using immature embryos of the wheat variety "Shi 4185" as explants, the control vector Ubi-GUS and Ubi-TaAGD vector were transformed by Agrobacterium-mediated method. Compared with the callus differentiation rate of 25.28% when the control vector Ubi-GUS was transformed, the callus differentiation rate of the overexpressing Ubi-TaAGD vector increased to 54.91% ( Figure 4 In terms of transformation efficiency, the overexpression control vector Ubi-GUS was 1.69%, while the overexpression vector Ubi-TaAGD increased to 31.21% ( Figure 5 In summary, overexpression of the TaAGD gene can effectively improve the genetic transformation efficiency of the difficult-to-transform wheat variety "Shi 4185". The TaAGD gene is a new gene that can improve the regeneration ability of wheat.

[0075] 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. Application of the TaAGD gene in improving the regeneration efficiency of wheat immature embryos; the TaAGD gene is a DNA molecule as shown in the following i) or ii): i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO. 2 except i).

2. Application of TaAGD protein in improving the regeneration efficiency of wheat immature embryos; the TaAGD protein is the protein 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) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

3. Application of recombinant expression vectors or genetically engineered bacteria containing the TaAGD gene in improving the efficiency of wheat immature embryo regeneration.

4. A method for improving the regeneration efficiency of wheat immature embryos, characterized in that: The following steps are involved: The TaAGD gene is connected to an expression vector to construct a recombinant expression vector, which is then transferred into Agrobacterium competent cells to obtain an Agrobacterium strain for transformation; and the Agrobacterium strain is used to infect wheat immature embryos.

5. The method according to claim 4, characterized in that The expression vector is pUbi110 vector; the TaAGD gene is connected between the SmaI and SpeI sites of the pUbi110 vector.

6. The method according to claim 4, characterized in that The Agrobacterium competent cells are Agrobacterium EHA105.

Images