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Application of TaDSK2a protein in regulating wheat resistance to stripe rust

A protein and rust technology, applied in the application field of TaDSK2a protein in regulating wheat resistance to stripe rust, can solve the problem of no TaUbiL function and its regulation method reported.

Active Publication Date: 2020-05-15
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Compared with humans, yeast and Arabidopsis, reports on DSK2 protein in wheat are still very rare. Kovalchuk et al. (Kovalchuk et al., 2009) used yeast two-hybrid to screen the interacting protein of TaPR60 and found that TaUbiL can interact with TaPR60 Signal peptide interaction, Bhowmik et al. (Bhowmik et al., 2018) used the CRISPR-Cas9 system to create TaUbiL mutants, but there was no further report on the function and regulation of TaUbiL

Method used

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  • Application of TaDSK2a protein in regulating wheat resistance to stripe rust
  • Application of TaDSK2a protein in regulating wheat resistance to stripe rust
  • Application of TaDSK2a protein in regulating wheat resistance to stripe rust

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Cloning of embodiment 1, TaDSK2a gene cDNA sequence

[0076] 1. Acquisition of wheat cDNA sequence

[0077] (1) Extraction of wheat total RNA

[0078] The total RNA of common wheat cultivar Suwon 11 was extracted with RNA extraction kit. The RNA extraction kit is a product of PEXBIO (Catalog No.: A010400), and the specific extraction steps and conditions are in accordance with the product instructions.

[0079] (2) Reverse transcription of RNA

[0080] The total RNA obtained in the above step (1) was reverse-transcribed using a reverse transcription kit (product number: RR047A) from Takara Company to obtain cDNA. The specific operation steps and conditions are required to be carried out in accordance with the product manual.

[0081] 2. PCR amplification and sequencing of TaDSK2a cDNA sequence

[0082] Using the cDNA obtained in the above step 1 as a template, using the primer pair DSKCDS-F and DSKCDS-R1, R2, the cDNA of wheat variety Suwon 11 (stripe rust resistan...

Embodiment 2

[0090] Embodiment 2, use BSMV-VIGS experiment to verify the anti-stripe rust function of TaDSK2a gene

[0091] 1. Obtaining of wheat with silenced TaDSK2a gene

[0092] 1. Construction of BSMV-VIGS vector system to induce TaDSK2a gene down-regulation

[0093] (1) According to the TaDSK2a-1D gene sequence (SEQ ID No.6) in Example 1, two silenced fragments at different positions were selected. One fragment was specifically located at +984bp~+1115bp, and the length was 132bp. It was named as the silent sequence TaDSK2a- 1. Obtained by PCR amplification of V-TaDSK-F1 and V-TaDSK.2-R1 using primers. The other fragment starts at +1279bp and ends at +1366bp, with a length of 88bp, named as the silent sequence TaDSK2a-2, obtained by PCR amplification using primer pair V-TaDSK-F2 and V-TaDSK-R2.

[0094] V-TaDSK-F1: 5'-CTA GCTAGC GCAAGGAACAACACGGTCA-3';

[0095] V-TaDSK-R1: 5'-CTA GCTAGC GCAGCACCAGTTCTTGCAT-3'.

[0096] V-TaDSK-F2: 5'-CTA GCTAGC CCAAATGCACGTAGCCTGA-3';

[00...

Embodiment 3

[0129] Example 3, using the Agrobacterium-mediated transgenic method to verify the anti-stripe rust function of the TaDSK2a gene

[0130] 1. Obtaining of TaDSK2a gene overexpression plants

[0131] 1. TaDSK2a gene overexpression vector primer design and vector construction

[0132] In order to verify the function of TaDSK2a gene in resistance to stripe rust in wheat, the present invention constructs TaDSK2a-1D gene into pLGY-02 vector driven by maize ubiquitin promoter (UBi1) (promoter contained in pLGY-02). The overexpression vector primers OETaDSK2a-F and OETaDSK2a-R were designed.

[0133] OETaDSK2a-F: 5'-CTAGAGGATCCCCG GGTACC -ATGGGCGGAGCAGGCGACGGCGAGG-3';

[0134] OETaDSK2a-R: 5'-GCTCTCTAGA ACTAGT -CTACTGGCCAAAGTTCCCGAGAAG-3'.

[0135] PCR reaction system: SEQ ID No.6 sequence product DNA obtained in Example 1 0.5 μL (<0.5 μg), dNTP (2.5 mM) 4 μl, OETaDSK2a-F (10 μM) 1 μl, OETaDSK2a-R (10 μM) 1 μl, 5 × TransStartFastPfuBuffer 10μl, TransStartFastPfu DNA Polymerase...

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Abstract

The invention discloses an application of a TaDSK2a protein in regulating wheat resistance to stripe rust. The invention provides an application of the TaDSK2a protein or a related biomaterial thereofin regulating plant resistance to stripe rust, wherein the related biomaterial is a nucleic acid molecule capable of expressing the TaDSK2a protein, or an expression cassette, recombinant vector, recombinant bacterium or transgenic cell line containing the nucleic acid molecule. The TaDSK2a gene can regulate wheat resistance to stripe rust. The application has important significance for cultivating wheat varieties with high resistance to stripe rust.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of TaDSK2a protein in regulating wheat resistance to stripe rust. Background technique [0002] Wheat stripe rust is a worldwide fungal disease caused by wheat stripe rust. Wheat stripe rust belongs to the Puccinia striiformis f.sp.tritici of the genus Puccinia, which is a living parasitic fungus. [0003] Stripe rust is a serious disease that endangers wheat production in China and the world. Although chemical agents can effectively control the economic losses caused by wheat diseases, the breeding and promotion of disease-resistant varieties is still considered to be the most fundamental, economical and safest way out of environmental safety and human health considerations (Line and Chen, 1995; Xu et al., 2013). The cloning and functional study of disease resistance genes are of great significance to the study of the molecular mechanism of wheat disease resistanc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29C07K14/415A01H5/00A01H6/46
CPCC07K14/415C12N15/8218C12N15/8282
Inventor 张相岐席海秀范仁春刘亚培沈前华
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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