Application of transcription factor TaMYBS2 in improving wheat resistance to stripe rust

Abstract

This invention discloses the application of the transcription factor TaMYBS2 in enhancing wheat resistance to stripe rust, belonging to the field of genetic engineering technology. This invention, through studying the function of the wheat transcription factor TaMYBS2 in its interaction with stripe rust fungi, discovered... TaMYBS2 The gene can respond to stripe rust infection and plays an important role in wheat disease resistance. Specifically, overexpression TaMYBS2 Genes can enhance wheat's resistance to stripe rust. Based on this, this invention constructs... TaMYBS2 Gene overexpression recombinant vectors were obtained, and Agrobacterium-mediated genetic transformation technology was used to obtain overexpressing genes. TaMYBS2 A transgenic wheat strain. This transgenic wheat strain exhibits significantly enhanced resistance to the stripe rust-affinity race CYR32, providing an important genetic resource for the creation of stripe rust-resistant wheat varieties.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to the application of transcription factor TaMYBS2 in improving wheat resistance to stripe rust. Background Technology

[0002] Wheat is an important food crop, produced by the wheat-specific strain of *Stripetra rust* (…). Puccinia striiformis f.sp. wheat , Psst Wheat stripe rust, caused by a fungal infection, is an airborne fungal disease that threatens the safe production of wheat. Breeding and planting resistant varieties is the most economical, effective, and environmentally friendly strategy for controlling wheat stripe rust. However, due to the frequent mutations in the virulence of stripe rust fungi, most varieties easily lose their rust resistance, and there is a lack of effective resistant resources in production. Therefore, discovering and utilizing new resistant genes and creating new stripe rust-resistant varieties are of great significance for improving wheat disease resistance and implementing green control measures.

[0003] Transcription factors are a class of proteins that bind to specific DNA sequences and regulate gene transcription initiation activity. By binding to cis-acting elements locally or distally to target genes, transcription factors activate or inhibit the expression of downstream genes, thereby participating in plant defense responses and stress signal transduction. Given the central role of transcription factors in plant growth, development, and disease resistance immunity, the rational utilization of their regulatory networks could potentially enhance plant disease resistance without affecting normal growth, while balancing the relationship between plant growth and development and disease resistance immunity. Plant MYB transcription factors play positive or negative regulatory roles in biotic stress responses. However, whether the wheat transcription factor TaMYBS2 responds to wheat stripe rust infection, and its specific function in the wheat-stripe rust interaction, remains unclear. Summary of the Invention

[0004] To address the aforementioned technical problems and deficiencies, this invention provides the application of transcription factor TamYBS2 in improving wheat resistance to stripe rust.

[0005] Firstly, this invention provides the application of the transcription factor TaMYBS2 in improving wheat resistance to stripe rust, through overexpression in wheat. TaMYBS2 Genes, or increasing the expression of the transcription factor Tamybs2, can enhance wheat's resistance to stripe rust;

[0006] The amino acid sequence of the transcription factor TaMYBS2 is shown in SEQ ID NO.1; The TaMYBS2 The open reading frame sequence of the gene is shown in SEQ ID NO.2.

[0007] Secondly, the present invention provides an overexpression recombinant vector for improving wheat stripe rust resistance, wherein the overexpression recombinant vector contains TaMYBS2 The open reading frame sequence of the gene, as shown in SEQ ID NO.2.

[0008] Thirdly, the present invention provides a method for improving wheat resistance to stripe rust, comprising: constructing TaMYBS2 Gene overexpression recombinant vectors were used to transfer overexpression recombinant vectors into wheat, and the resulting overexpressing genes were screened. TaMYBS2 Genetically modified wheat.

[0009] Furthermore, in the method for improving wheat resistance to stripe rust provided by the present invention, the primers for constructing the overexpression recombinant vector are shown in SEQ ID NO. 3~4.

[0010] Furthermore, in the method for improving wheat resistance to stripe rust provided by the present invention, the blank vector for constructing the overexpression recombinant vector is pANIC6E.

[0011] Furthermore, in the method for improving wheat resistance to stripe rust provided by the present invention, the method for transferring the overexpression recombinant vector into wheat is Agrobacterium-mediated genetic transformation.

[0012] Furthermore, in the method for improving wheat resistance to stripe rust provided by the present invention, the overexpression recombinant vector contains a promoter, a terminator, and a selection marker gene.

[0013] Furthermore, in the method for improving wheat resistance to stripe rust provided by the present invention, the screening marker gene is the phosphinic acid acetyltransferase gene.

[0014] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: This invention reveals the key role of transcription factor TaMYBS2 in wheat resistance to stripe rust and successfully creates new wheat germplasm with high resistance to stripe rust using transgenic technology, which is of great significance for breeding stable and genetically resistant wheat varieties.

[0015] This invention also provides a method for breeding wheat varieties resistant to stripe rust, comprising the following steps: constructing a wheat variety containing... TaMYBS2 The gene overexpression recombinant vector was transferred into wheat immature embryos using Agrobacterium-mediated genetic transformation to obtain overexpressed genes. TaMYBS2 Transgenic wheat with genetically modified genes. Resistance testing showed that the transgenic wheat obtained using the method of this invention exhibited significantly enhanced resistance to stripe rust, effectively enriching existing disease-resistant germplasm resources. Attached Figure Description

[0016] Figure 1 Induced by stripe rust fungi TaMYBS2 Gene expression status. Among them, (A) is the incompatible race CYR23 of stripe rust; (B) is the compatible race CYR32 of stripe rust.

[0017] Figure 2 for TaMYBS2 A schematic diagram of the structure of a gene overexpression recombinant vector.

[0018] Figure 3 For overexpression TaMYBS2 PCR test results of genetically modified wheat.

[0019] Figure 4 For overexpression TaMYBS2 Expression analysis of the gene in transgenic wheat and phenotypic results of inoculation with the stripe rust-compatible race CYR32. (A) shows the expression level analysis, and (B) shows the phenotypic results. Detailed Implementation

[0020] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially.

[0021] The amino acid sequence (SEQ ID NO.1) of the TaMYBS2 protein involved in the following examples is as follows: MEEEGARKAVLFRLFGVEVRGAEEEDDAEPMELKKSTSMPNLACASNSPPLLLPGEASHDKGYASDDGELASTPQLKRCRRKAQERKKGIPWTEEEHRKFLEGLKQLGKGDWRGISKNFVTTRTATQVASHAQKYFLRQTNPGKKKRRASLFDVGIPAAHSYDDQLPSPQSVGTKLAPAEKILHTDRGDVPLPSYPGGIRGSNNNMQMQADELTDRVKKRSKFPTGTSLAAMAASGLELAMSSSASSILELSIAPPRCYGAVDAIKVL; The matters involved TaMYBS2The open reading frame sequence of the gene (SEQ ID NO.2) is as follows: ATGGAGGAGGAGGGCGCGAGGAAGGCGGTGCTCTTCCGGCTGTTCGGCGTCGAGGTCCGCGGCGCGGAGGAGGAGGATGACGCGGAGCCCATGGAGCTCAAGAAGAGCACCAGCATGCCCAACCTCGCCTGCGCCTCCAACAGCCCGCCGCTTCTCCTGCCCGGCGAGGCCAGCCACGACAAGGGCTACGCCTCCGACGACGGCGAGCTGGCGTCCACGCCGCAGCTCAAGCGCTGCCGCCGCAAGGCCCAGGAGCGCAAGAAAGGGATTCCGTGGACCGAGGAGGAGCACAGGAAGTTCCTGGAGGGTCTGAAGCAGCTGGGCAAGGGGGACTGGAGAGGCATCTCCAAGAACTTCGTCACCACCAGGACGGCCACGCAGGTGGCTAGCCACGCCCAGAAGTACTTCCTCCGGCAGACCAACCCCGGCAAGAAGAAGCGCCGGGCCAGCCTCTTCGACGTCGGCATCCCCGCTGCCCACAGCTACGACGATCAGCTGCCAAGTCCTCAGAGCGTTGGAACCAAGCTTGCTCCTGCTGAGAAGATACTCCACACAGACCGCGGCGACGTGCCGCTACCAAGTTATCCTGGAGGCATCAGGGGCAGCAACAACAACATGCAGATGCAGGCTGATGAGCTAACTGACCGTGTGAAGAAGAGATCAAAGTTCCCCACCGGGACGTCGCTGGCCGCCATGGCTGCCTCCGGGCTGGAGCTGGCAATGTCTTCGTCTGCCAGCAGCATCCTGGAGCTCAGCATCGCGCCTCCTCGCTGCTACGGCGCGGTCGACGCCATCAAGGTGCTGTGA。

[0022] Example 1 This example provides TaMYBS2 the amino acid sequence of the wheat

[0023] This example obtains the TaMYBS2 sequence from the NCBI website (https: / / www.ncbi.nlm.nih.gov / books / ). Design TaMYBS2 Using specific amplification primers and cDNA from Fielder plants (provided by the Plant Immunology Laboratory of Northwest A&F University) as a template, the amplification yielded... TaMYBS2 Open reading frame (ORF) sequences.

[0024] TaMYBS2 The ORF sequence is shown in SEQ ID NO.2, and its amino acid sequence is shown in SEQ ID NO.1.

[0025] Amplification TaMYBS2 The primers for the gene are as follows: TaMYBS2 -F (SEQ ID NO.3): 5'-ATGGAGGAGGAGGGCGCGA-3'; TaMYBS2 -R (SEQ ID NO. 4): 5'-TCACAGCACCTTGATGGC-3'.

[0026] Example 2 This embodiment provides wheat TaMYBS2 Expression of genes during interactions with stripe rust fungi.

[0027] When wheat (Water Source 11) reached the two-leaf-one-heart stage, the incompatible race CYR23 and compatible race CYR32 of stripe rust fungus were inoculated onto wheat leaves using a brush application method. The specific procedure included: taking fresh stripe rust spores into centrifuge tubes, adding electronic fluorination solution to obtain spore suspensions, and using a clean, dry brush to apply a small amount of spore suspension gently and evenly to the upper surface of the two wheat leaves. After inoculation, the wheat was placed in a 16 ℃ dark environment for 24 h to maintain humidity, then transferred to a 16 ℃ constant temperature and light incubator (16 h light / 8 h dark) for normal culture. RNA was extracted from wheat leaves at 0, 6, 12, 24, 48, 72, 96, and 120 h post-inoculation and reverse transcribed. Wheat elongation factor was then used as the RNA. TaEF-α As an internal reference gene, 2 –ΔΔCt Relative quantification algorithms, through real-time quantitative PCR analysis TaMYBS2 Gene expression at different time points after wheat infection with stripe rust fungus. Primers for real-time quantitative PCR analysis are as follows: TaEF-α -qRT-F (SEQ ID NO.5): 5'-TGGTGTCATCAAGCCTGGTATGGT-3'; TaEF-α-qRT-R (SEQ ID NO. 6): 5'-ACTCATGGTGCATCTCAACGGACT-3'.

[0028] TaMYBS2 -qRT-F (SEQ ID NO.7): 5'-CAACATCCAGATGCAGGTTGAA-3'; TaMYBS2 -qRT-R (SEQ ID NO. 8): 5'-CAGATGAAGACATTGCCAGCTC-3'.

[0029] like Figure 1 As shown, wheat at 24 h and 48 h after inoculation with the incompatible race CYR23 of stripe rust, TaMYBS2 Gene expression was significantly upregulated; wheat inoculated with the stripe rust-affinity race CYR32 at 24 h, 48 h, and 72 h, TaMYBS2 The gene was significantly induced to express, indicating TaMYBS2 Genes can respond to stripe rust infection and play an important role in the process of stripe rust infection of wheat.

[0030] Example 2 This embodiment provides TaMYBS2 Construction of gene overexpression recombinant vectors and their introduction into wheat.

[0031] Using cDNA from Fielder plants as a template, TaMYBS2 Primers for gene overexpression recombinant vectors were used for PCR amplification to obtain the target fragment. This target fragment was then ligated into the blank overexpression recombinant vector pANIC6E via a Gateway reaction to construct the desired gene overexpression vector. TaMYBS2 Gene overexpression recombinant vectors, such as Figure 2 As shown in the figure. LB and RB are homologous arms; Bar is the phosphinic acid acetyltransferase gene, used as a herbicide selection marker gene; ZmUbi is the promoter; GUSPlus is the β-glucuronidase reporter gene; and T-NOS represents the terminator. TaMYBS2 The primers for the gene overexpression recombinant vector are as follows: TaMYBS2 -6E-F (SEQ ID NO.9): 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTACCCATACGACGTCCCAGACTACGCTATGGAGGAGGAGGGCGCGA-3'; TaMYBS2-6E-R (SEQ ID NO. 10): 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAAGCGTAGTCTGGGACGTCGTATGG GTACAGCACCTTGATGGC-3'.

[0032] The constructed TaMYBS2 The overexpression recombinant vector was added to Agrobacterium competent cells EHA105, and then placed on ice for 5 min, followed by liquid nitrogen for 5 min, a 37°C water bath for 5 min, and then on ice for 5 min again. 700 μL of antibiotic-free liquid LB medium was then added, and the cells were cultured at 28°C with shaking at 200 rpm for 2 h. The cells were then collected by centrifugation at 5000 rpm for 1 min at room temperature. The supernatant was discarded, and 100 μL of the supernatant was used to resuspend the cells. The cells were then plated onto LB agar plates containing 50 μg / mL kanamycin and 25 μg / mL rifampicin. The plates were inverted and incubated at 28°C for 2–3 days until single colonies appeared. Using the transgenic platform of the State Key Laboratory of Arid Zone Crop Stress Biology, Agrobacterium-mediated genetic transformation technology was employed to transform the cells into... TaMYBS2 The pANIC6E plasmid was transferred into the immature embryos of the wheat variety "Fielder" and overexpressed. TaMYBS2 Transgenic wheat T0 generation regenerated seedlings were screened for positive plants by PCR detection.

[0033] Figure 3 PCR test results showed that, with overexpression TaMYBS2 Transgenic wheat lines TaMYBS2 -OE-L85 and TaMYBS2 Using the genomic DNA of -OE-L94 as a template, a specific band of the same size as the positive control (overexpression recombinant vector, Plasmid) was amplified, while wild-type wheat (Fielder) did not show this band. These results indicate that... TaMYBS2 The gene overexpression recombinant vector was successfully constructed and contains the complete target gene. TaMYBS2 The gene has been successfully integrated into the genomes of two transgenic wheat lines.

[0034] T1 generation genetically modified wheat TaMYBS2 OE-L85 TaMYBS2When OE-L94 and wild-type wheat (Fielder) reached the two-leaf-one-heart stage, fresh stripe rust spores (a related race of stripe rust, CYR32) were suspended in an electronically fluorinated solution to obtain a spore suspension. The spore suspension was evenly applied to the upper surface of the two leaves using a brush. After inoculation, the plants were placed in a 16°C environment and incubated under moist conditions for 24 hours. Then, they were transferred to a 16°C constant temperature and light incubator (16 hours of light / 8 hours of darkness) for normal incubation. Sporulation on the wheat leaves was observed after 14 days.

[0035] Figure 4 (A) in the text represents genetically modified wheat. TaMYBS2 OE-L85 TaMYBS2 The expression levels of OE-L94 and wild-type wheat (Fielder) were detected, proving that transgenic wheat... TaMYBS2 OE-L85 TaMYBS2 All of these have been implemented in OE-L94. TaMYBS2 Gene overexpression. For example... Figure 4 As shown in (B), compared to wild-type wheat (Fielder), genetically modified wheat... TaMYBS2 OE-L85 and TaMYBS2 No obvious urediniospores or obvious necrosis were observed in the leaves of OE-L94, indicating that the transgenic wheat... TaMYBS2 OE-L85 and TaMYBS2 OE-L94 exhibits good resistance to stripe rust fungi. These results indicate that overexpression of OE-L94 in wheat... TaMYBS2 Genes can enhance wheat's resistance to stripe rust.

[0036] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. The application of transcription factor TaMYBS2 in improving wheat resistance to stripe rust, characterized in that, Overexpression in wheat TaMYBS2 Genes, or increasing the expression of the transcription factor Tamybs2, can enhance wheat's resistance to stripe rust; The amino acid sequence of the transcription factor TaMYBS2 is shown in SEQ ID NO.1; The TaMYBS2 The open reading frame sequence of the gene is shown in SEQ ID NO.

2.

2. A method for improving wheat resistance to stripe rust, characterized in that, include: Build TaMYBS2 Gene overexpression recombinant vectors were used to transfer overexpression recombinant vectors into wheat, and the resulting overexpressing genes were screened. TaMYBS2 Genetically modified wheat.

3. The method for improving wheat resistance to stripe rust according to claim 2, characterized in that, The primers for constructing the overexpression recombinant vector are shown in SEQ ID NO.3~4.

4. The method for improving wheat resistance to stripe rust according to claim 2, characterized in that, The blank vector used to construct the overexpression recombinant vector was pANIC6E.

5. The method for improving wheat resistance to stripe rust according to claim 2, characterized in that, The method for transferring the overexpression recombinant vector into wheat is Agrobacterium-mediated genetic transformation.

6. The method for improving wheat resistance to stripe rust according to claim 2, characterized in that, The overexpression recombinant vector contains a promoter, a terminator, and a selection marker gene.

7. The method for improving wheat resistance to stripe rust according to claim 6, characterized in that, The screening marker gene is the phosphinic acid acetyltransferase gene.

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