Gene for improving wheat immature embryo transformation efficiency and application thereof

Abstract

The invention discloses a gene for improving wheat immature embryo transformation efficiency and application thereof, and belongs to the technical field of plant genetic engineering. According to the invention, a new wheat regeneration gene TaDJA gene is found, and the TaDJA gene is over-expressed in a wheat variety 'Kefeng No.3' which is extremely difficult to transform, so that the callus induction rate and the transformation efficiency of wheat immature embryos can be improved; the method is beneficial for integrating exogenous nucleic acid molecules into the genome of the wheat variety difficult to transform, and provides technical support for genetic improvement of the wheat variety difficult to transform.

Description

Technical Field

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

[0002] Wheat is one of the most important food crops in the world. It not only provides carbohydrates, protein and dietary fiber for humans, but also has a wide range of uses in food processing, feed and industrial raw materials. However, with global climate change, population growth and shrinking arable land resources, wheat production faces severe challenges, such as the threats of biotic and abiotic stresses such as pests and diseases, drought and high temperature. To meet these challenges, modern biotechnology and gene editing will become important methods for wheat breeding. The low genetic transformation efficiency of wheat is the main bottleneck restricting biological breeding, while the low regeneration ability and strong genotype dependence make many excellent genes difficult to apply in actual breeding. Therefore, using regeneration-related genes to enhance the regeneration and genetic transformation ability of wheat can accelerate the verification of gene function and provide excellent genetic resources for the breeding of higher-yielding, high-quality and stress-resistant wheat varieties, thereby ensuring global food security and promoting sustainable agricultural development.

[0003] DJA protein is an important member of the plant HSP40 family and is mainly involved in processes such as protein folding, degradation, and stress response. Studies have shown that DJA protein plays a key role in plant adaptation to stress conditions such as cold, heat, and drought, and regulates gene expression during developmental stages such as seed germination and flowering. This protein works synergistically with molecular chaperones such as HSP70 to participate in regulating complex protein functional networks and maintain cell homeostasis. In Arabidopsis, the AtDjA3 gene plays a key role in the tolerance of abiotic stress ( Set al., 2016). The rice OsDjA6 gene mediates immune responses against rice blast by participating in the salicylic acid signaling pathway (Yang Jiuxia, 2015). However, research on the functions of DJA proteins in wheat is relatively limited, and their role in promoting wheat regeneration and genetic transformation has not been reported. Summary of the Invention

[0004] In view of the above-mentioned prior art, the object of the present invention is to provide a gene for improving the transformation efficiency of wheat immature embryos and its application.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides the use of the TaDJA gene in the following (1) or (2):

[0007] (1) Improve the callus induction rate of wheat immature embryos;

[0008] (2) Improve the transformation efficiency of wheat immature embryos;

[0009] The TaDJA gene is a nucleic acid molecule as shown in the following i) or ii):

[0010] i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1;

[0011] ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO. 2 except i).

[0012] In the above application, the wheat is a wheat variety that is difficult to transform, for example, "Kefeng No. 3" wheat.

[0013] In the above application, the callus induction rate and transformation efficiency of wheat immature embryos can be improved by overexpressing the TaDJA gene.

[0014] In the above application, the transformation efficiency refers to the ratio of the number of positive seedlings obtained after genetic manipulation of wheat immature embryos to the total number of immature embryos.

[0015] The second aspect of the present invention provides the use of TaDJA protein in the following (1) or (2):

[0016] (1) Improve the callus induction rate of wheat immature embryos;

[0017] (2) Improve the transformation efficiency of wheat immature embryos.

[0018] Preferably, the TaDJA protein is the protein shown in (A1) or (A2) below:

[0019] (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO. 2 in the sequence listing;

[0020] (A2) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0021] 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).

[0022] The third aspect of the present invention provides the use of an expression cassette, a recombinant expression vector or a recombinant bacterium containing the TaDJA gene in improving wheat transformation efficiency.

[0023] A fourth aspect of the present invention provides a method for improving wheat transformation efficiency, comprising the following steps:

[0024] The TaDJA 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.

[0025] Preferably, the expression vector is pUbi110 vector.

[0026] Preferably, the wheat immature embryos are immature embryos of "Kefeng No. 3" wheat 14-15 days after pollination.

[0027] Beneficial effects of the present invention:

[0028] The present invention discovered a new wheat regeneration gene, TaDJA. Overexpressing the TaDJA gene in the extremely difficult-to-transform wheat variety "Kefeng No. 3" can improve the callus induction rate and transformation efficiency of wheat immature embryos; it is beneficial to integrate exogenous nucleic acid molecules into the genome of difficult-to-transform wheat varieties, and provides technical support for the genetic improvement of difficult-to-transform wheat varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the characteristics of the plant expression vector Ubi-TaDJA;

[0030] Figure 2 Schematic diagram of the structure of the plant expression vector Ubi-GUS;

[0031] Figure 3 Schematic diagram of PCR-specific amplification of the bar gene in candidate transgenic plants transformed with the plant expression vector Ubi-TaDJA. M represents a 2000 bp molecular weight marker, 1-13 represent candidate transgenic plants transformed with Kefeng 3, PC represents the positive plasmid, NC represents the negative control, and WT represents the wild-type control.

[0032] Figure 4 The callus induction rate of immature embryos of wheat variety Kefeng 3 infected with plant expression vector Ubi-TaDJA and control vector Ubi-GUS.

[0033] Figure 5 The transformation efficiency of the plant expression vector Ubi-TaDJA and the control vector Ubi-GUS in infecting the immature embryos of the wheat variety Kefeng No. 3. DETAILED DESCRIPTION

[0034] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0035] As mentioned earlier, low genetic transformation efficiency in wheat is a major bottleneck limiting wheat biotechnology breeding. Low regeneration capacity and strong genotype dependence make many high-quality genes difficult to apply in practical breeding. "Kefeng No. 3" is a superior spring wheat germplasm resource developed through multi-hybridization at the Keshan Agricultural Science Institute of the Heilongjiang Academy of Agricultural Sciences. Due to its excellent overall traits, "Kefeng No. 3" has been used as a parent for many years and currently requires further genetic improvement of its traits. However, "Kefeng No. 3" is an extremely difficult wheat variety to transform, making it difficult to genetically improve it through the introduction of exogenous genes through transgenic methods.

[0036] In view of this, the present invention used "Kefeng No. 3" as the experimental object and conducted in-depth research on how to improve its transformation efficiency. The TaDJA gene is a member of the DJA protein family, and its nucleotide sequence is shown in SEQ ID NO.1, which is as follows:

[0037]

[0038] The amino acid sequence of the TaDJA protein encoded by the TaDJA gene is shown in SEQ ID NO. 2, and is as follows:

[0039] MFGRMPRKTSNNTKYYEVLGVSKTATPDELKKAYRKAAIKNHPDKGGDPEKFKELAQAYDVLNDPEKREIYDQYGEDAIKEGMGGSGGADMHSPFDIFEQLFGGGG GGGFGGSSRGRRQKRGEDVVHTMKVSLEDLYNGATKKLSLSRNVLCGKCKGKGSKSGATATCSGCRGAGMRMITRQIGPGMIQQMNTVCPECRGSGEMINDKDRCPS CRGNKVSQEKKVLEVHVEKGMQHGQKIVFQGEADEAPDTVTGDIVFVLQLKEHPKFKRKSDDLFVEHTISLTEALCGFQFVLTHLDGRQLLIKSNPGEIIKPGQHK AINDEGMPQHGRPFMKGRLFVEFSVEFPEPGVLTPSQCKSLEKILPPRPGSQSSDMDVDQCEETTMHDVNIEEEMRRRQHQRRQEAYDEEDDDEGGAPRGVQCAQQ.

[0040] Currently, there are few reports on the function of the TaDJA gene. The present invention discovered that the TaDJA gene can be used as a regeneration gene to improve the transformation efficiency of "Kefeng No. 3". Overexpressing the TaDJA gene in "Kefeng No. 3" can significantly improve the callus induction rate and transformation efficiency of "Kefeng No. 3", thus proposing the present invention.

[0041] 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.

[0042] 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.

[0043] The culture medium components used in the embodiments of the present invention are as follows:

[0044] WLS-AS medium: 1 / 10 MS minimal medium, 1 / 10 MS vitamins, glucose 10 g / L, acetosyringone 100 μM, agarose 8 g / L.

[0045] 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.

[0046] WLS-P5 medium: WLS-Res medium supplemented with PPT 5 mg / L.

[0047] WLS-P10 medium: WLS-Res medium supplemented with 10 mg / L PPT.

[0048] LSZ-P5 medium: MS basic medium, LS vitamins, zeatin 5 mg / L, sucrose 20 g / L, carbenicillin 250 mg / L, PPT 5 mg / L, phytogenin 3 g / L.

[0049] LSF-P5 medium: MS basic medium, LS vitamins, IBA 0.2 mg / L, sucrose 15 g / L, carbenicillin 250 mg / L, PPT 5 mg / L, and phytogenin 3 g / L.

[0050] Example 1: Construction of plant expression vector Ubi-TaDJA:

[0051] Based on the TaDJA gene sequence, specific primers were designed and the full-length CDS sequence of the gene was successfully cloned. The corresponding nucleotide sequence is shown in SEQ ID NO. 1 and is named TaDJA. The primers required for gene amplification and overexpression vector construction are shown in Table 1.

[0052] Table 1: Primer sequences for TaDJA gene cloning and overexpression vector construction

[0053] Primer name Sequence (5'-3') use DJA-F1 AGGAAGCCCATTTACCAGCC Gene amplification DJA-R1 AGTGCGCCCAGCAGTAA Gene amplification DJA-110F CGACTCTAGAGGATCCCCGGGATGTTTGGACGCATGCCAA Homology arm amplification DJA-110R GAATTCCGGCTCGAGACTAGTTTACTGCTGGGCGCACTGC Homology arm amplification 110-1F GCTCTAACCTTGAGTACCTA PCR screening DJA-J-R1 CCGCTCTTGGATCCCTTTC PCR screening

[0054] Reference The amplified product was recovered and ligated to the Blunt3 vector using a Cloning Kit (Beijing Quanshijin Biotechnology Co., Ltd., Catalog No.: CB301-01). The product was transformed into Escherichia coli DH5α, and single clones were picked for sequencing. The correct single clones were extracted with the FastPure Plasmid Mini Kit (Nanjing Novogene Biotechnology Co., Ltd., Catalog No.: DC201-01) to prepare the pEASY-Blunt3-TaDJA plasmid.

[0055] After sequencing analysis, the nucleotide sequence of the PCR amplification product is Sequence 1 in the sequence list, and the gene shown by the PCR product is named TaDJA gene; the protein encoded by the gene is named TaDJA, and the amino acid sequence of the protein is Sequence 2 in the sequence list.

[0056] Using pEASY-Blunt3-TaDJA plasmid as template, PCR amplification was performed using homologous recombination primers. TM DNA Assembly Mix Plus (Jiangsu Yugong Life Science Co., Ltd.) was used for homologous recombination and ligated with the pUbi110 vector digestion product for sequencing. The correct single clone was extracted with the FastPurePlasmid Mini Kit (Nanjing Novozymes Biotech Co., Ltd., Cat. No. DC201-01) to obtain Ubi-TaDJA. The schematic diagram of the vector structure is shown in the figure. Figure 1 shown.

[0057] Example 2: Construction of plant expression vector Ubi-GUS:

[0058] 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., catalog number: P515). The primer sequences are shown in Table 2.

[0059] Table 2: Primer sequences for GUS gene cloning and vector construction

[0060] Primer name Sequence (5'-3') use GUS-F ATGTTACGTCCTGTAGAA Gene amplification GUS-R TCATTGTTTGCCTCCCTG Gene amplification GUS-110F CGACTCTAGAGGATCCCCGGGATGTTACGTCCTGTAGAAACCCCA Homology arm amplification GUS-110R GAATTCCGGCTCGAGACTAGTTTGTTTTGCCTCCCTGCTGC Homology arm amplification 110-1F GCTCTAACCTTGAGTACCTA PCR screening GUS-J-R1 GCGTCGCAGAACATTACA PCR screening

[0061] Reference The amplified product was recovered and ligated to the Blunt3 vector, and then transformed into Escherichia coli DH5α. Single clones were picked for sequencing, and the correct single clones were sequenced using the FastPure Plasmid Mini Kit (Nanjing Novogene Biotech Co., Ltd., Catalog No.: DC201-01) to extract the plasmid, and the pEASY-Blunt3-GUS plasmid was prepared.

[0062] Using pEASY-Blunt3-GUS plasmid as template, homologous recombination primers were designed for PCR amplification. TM DNA Assembly Mix Plus (Jiangsu Yugong Life Science Co., Ltd.) was used for homologous recombination and ligated with the pUbi110 vector digestion product for sequencing. The correct single clone was extracted with the FastPurePlasmid Mini Kit (Nanjing Novozymes Biotech Co., Ltd., Cat. No. DC201-01) to obtain Ubi-GUS. The schematic diagram of the vector structure is shown in the figure. Figure 2 shown.

[0063] Example 3: Obtaining TaDJA gene overexpressing transgenic wheat and investigating the transformation effect

[0064] 1. Preparation of recombinant Agrobacterium:

[0065] The Ubi-TaDJA prepared in Example 1 was transformed into Agrobacterium EHA105 competent cells, and an Agrobacterium strain suitable for transformation was obtained, which was named Ubi-TaDJA / EHA105.

[0066] The Ubi-GUS prepared in Example 2 was transformed into Agrobacterium EHA105 competent cells, and an Agrobacterium strain suitable for transformation was obtained, which was named Ubi-GUS / EHA105.

[0067] 2. Agrobacterium-mediated transformation of wheat immature embryos:

[0068] The detailed steps and methods of the Agrobacterium-mediated method for wheat embryos were referred to the method of Ishida et al. (Ishida et al., 2015), as follows:

[0069] (1) Three days before infection, Agrobacterium tumefaciens (Ubi-TaDJA / EHA105) and Agrobacterium tumefaciens (Ubi-GUS / EHA105) were inoculated onto YEP solid culture 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 culture 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 Agrobacterium tumefaciens resuspensions of Ubi-TaDJA / EHA105 and Ubi-GUS / EHA105, respectively.

[0070] (2) The immature embryos of wheat "Kefeng No. 3" about 2 weeks after pollination were collected and infected with Agrobacterium resuspensions of Ubi-TaDJA / EHA105 and Ubi-GUS / EHA105, respectively. The embryos were spread on WLS-AS medium with the scutellum facing upward and cultured in a dark incubator at 23°C for 2 days.

[0071] (3) The co-cultured embryos were transferred to WLS-Res medium and cultured in a dark incubator at 25°C for 5 days.

[0072] (4) The callus tissue after recovery culture was transferred to WLS-P5 medium and cultured in a dark incubator at 25°C for 2 weeks.

[0073] (5) The callus tissue was then transferred to WLS-P10 medium and cultured in a dark incubator at 25°C for 3 weeks.

[0074] (6) The above callus tissue was transferred to LSZ-P5 medium and cultured in a 25°C incubator under light for 2 weeks.

[0075] (7) The regenerated resistant buds of wheat were transferred to LSF-P5 medium and cultured in a 25°C incubator under light until the roots of the regenerated buds were about 1-2 cm long.

[0076] (8) The rooted and strong seedlings were transplanted into nutrient soil to obtain candidate transgenic seedlings of Ubi-TaDJA and Ubi-GUS, respectively.

[0077] 3. PCR detection of candidate transgenic plants:

[0078] 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-TaDJA / EHA105 and Ubi-GUS / EHA105 vectors, respectively.

[0079] The extracted genomic DNA of the candidate transgenic plants was used as a template for PCR amplification using TaqDNA polymerase (Nanjing Novozymes Biotech Co., Ltd., Catalog No.: P222). The primer sequences were:

[0080] bar-F: GGCGGTCTGCACCATCGTCAACCACTAC;

[0081] bar-R: AGTCCAGCTGCCAGAAACCCACGTCATG.

[0082] PCR identification results Figure 3 As shown, a 446 bp bar gene fragment was amplified in candidate transgenic plants 1-13, all of which were positive plants, while the negative control and wild-type wheat did not have a 446 bp bar gene fragment.

[0083] 4. Wheat conversion efficiency statistics:

[0084] Immature embryos of the wheat variety Kefeng No. 3 were collected 14-15 days after pollination and infected with Agrobacterium-mediated infection to induce callus formation. The calli were then transferred to a screening medium for culture and differentiation into resistant seedlings. The number of immature embryos and calli were counted on LSF-P5 medium. After PCR identification, the number of transgenic seedlings was counted, and the callus induction rate ((number of calli ÷ total number of immature embryos) × 100%) and transformation efficiency ((number of positive seedlings ÷ total number of immature embryos) × 100%) were calculated.

[0085] Using immature embryos of the wheat variety Kefeng 3 as explants, the Ubi-GUS control vector and the Ubi-TaDJA vector were transformed using Agrobacterium-mediated method. Compared with the control vector Ubi-GUS, the callus induction rate of the Ubi-TaDJA vector was increased from 75.59% to 88.89% ( Figure 4 ), the transformation efficiency was increased from no transformation seedlings to 20.60% ( Figure 5 The above results indicate that the TaDJA gene can effectively improve the callus induction rate and transformation efficiency of Kefeng 3, and is a new gene that can improve the regeneration ability of wheat, providing important genetic resources and technical support for wheat genetic improvement.

[0086] 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 TaDJA gene in (1) or (2): (1) Improve the callus induction rate of wheat immature embryos; (2) Improve the transformation efficiency of wheat immature embryos; The TaDJA gene is a nucleic acid 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. The use according to claim 1, characterized in that The wheat is a wheat variety that is difficult to transform.

3. The use according to claim 2, characterized in that The wheat described is "Kefeng No. 3" wheat.

4. Use of TaDJA protein in the following (1) or (2): (1) Improve the callus induction rate of wheat immature embryos; (2) Improve the transformation efficiency of wheat immature embryos.

5. The use according to claim 4, characterized in that The TaDJA protein is a protein 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) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

6. Application of expression cassettes, recombinant expression vectors or recombinant bacteria containing the TaDJA gene in improving wheat transformation efficiency.

7. A method for improving wheat transformation efficiency, characterized in that: The following steps are involved: The TaDJA 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.

8. The method according to claim 7, characterized in that The expression vector is pUbi110 vector.

9. The method according to claim 7, characterized in that The wheat immature embryos are immature embryos of "Kefeng No. 3" wheat 14-15 days after pollination.

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