Wheat disease resistance protein taafrk and its related biomaterials and applications

A biomaterial and protein technology, applied in wheat disease-resistant protein TaAFRK and its related biomaterials and applications, can solve the problems of slow progress in breeding of wheat disease-resistant varieties, difficulty in field identification, and shortage

Active Publication Date: 2022-03-15
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of readily available wheat germplasm resources with high resistance to the above soil-borne fungal diseases, field identification is difficult, making the progress of conventional breeding methods to breed wheat disease-resistant varieties slow

Method used

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  • Wheat disease resistance protein taafrk and its related biomaterials and applications
  • Wheat disease resistance protein taafrk and its related biomaterials and applications
  • Wheat disease resistance protein taafrk and its related biomaterials and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Embodiment 1, the cloning of wheat disease resistance protein TaAFRK and its coding gene

[0101] 1. Cloning of TaAFRK gene

[0102] The inventors of the present invention isolated and cloned a wheat disease resistance-related protein from sheath blight resistant wheat germplasm CI12633, the amino acid sequence of which is shown in SEQ ID No.2, and named it TaAFRK protein. The gene encoding the TaAFRK protein is named TaAFRK gene, as shown in SEQ ID No.1, and the specific cloning method is as follows:

[0103] Total RNA was extracted from the stems of wheat CI12633 inoculated with wheat sheath blight (Rhizoctonia graminearum strain WK207), and the extracted RNA samples were reverse-transcribed to synthesize the first cDNA Strand cDNA was used as a template for gene cloning, using TaAFRK-OF and TaAFRK-OR as primers, and TOYOBO's high-fidelity TAQ enzyme (KOD FX), KOD FX Buffer, and dNTPs for PCR amplification.

[0104] TaAFRK-OF: 5'-CGCGGTTAGGTAGCTAGCAT-3';

[0105] T...

Embodiment 2

[0122] Example 2, TaAFRK gene expression level is closely related to wheat sheath blight resistance

[0123] 1. Analysis of the expression characteristics of TaAFRK gene in different varieties

[0124] Inoculate wheat sheath blight bacteria (Rhizoctonia graminearum WK207) for two days, and take sheath blight-resistant wheat varieties / lines (CI12633, Shanhong wheat), moderately resistant wheat varieties (Shannong 0431 and Xifeng) and susceptible wheat Wheat leaf sheaths and stem tissues at the inoculation sites of varieties (Wenmai No. 6 and Yangmai No. 9) were quick-frozen in liquid nitrogen. The RNA of the above six wheat materials was extracted with TRIZOL and purified.

[0125] About 5 μg of total RNA from each sample was reverse-transcribed into cDNA according to the procedure of the first-strand cDNA synthesis kit of Tiangen Biochemical Company. The cDNA concentrations of the samples were normalized using the constitutively expressed actin gene as an internal reference....

Embodiment 3

[0130] Example 3. Reverse verification of TaAFRK gene function by cultivating wheat with reduced resistance to sheath blight

[0131] 1. Silencing the TaAFRK gene in wheat CI12633 using virus-mediated gene silencing technology

[0132] 1. The two ends of the DNA fragment shown in nucleotides 1588-1817 of SEQ ID No.1 (ie, SEQ ID No.3) are respectively provided with NheI recognition sequences. After NheI digestion, the DNA fragment (230bp) shown in the 1588-1817th nucleotide of SEQ ID No.1 (ie SEQID No.3) is inserted into the NheI digestion of the BSMV-γ chain by reverse insertion site, to obtain the recombinant vector BSMV-γ: antiTaAFRK, so that the DNA molecule (antiTaAFRK) reverse complementary to the DNA fragment shown in the 1588-1817 positions (ie SEQ ID No.3) of SEQ ID No.1 is γ The vector is driven by a T7 promoter.

[0133] 2. Preparation of transcription reaction solution

[0134] (1) Take the BSMV-α plasmid, digest it with the restriction endonuclease MluI, recover...

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Abstract

The invention discloses wheat disease resistance protein TaAFRK and related biological materials and applications. TaAFRK is any of the following proteins: A1) a protein whose amino acid sequence is as shown in SEQ ID No.2; A2) the amino acid sequence defined by A1) undergoes substitution and / or deletion and / or addition of one or several amino acid residues The obtained protein has more than 90% identity with the protein shown in A1) and is related to plant disease resistance; A3) A fusion protein obtained by linking tags at the N-terminal or / and C-terminal of A1) or A2). The TaAFRK and its encoding gene provided by the invention can be used as a fungal inhibitor and for improving plant disease resistance, and are of great significance for cultivating new plant disease-resistant varieties.

Description

technical field [0001] The invention relates to wheat disease-resistant protein TaAFRK and its related biological materials and applications in the field of biotechnology. Background technique [0002] Wheat is one of the most important food crops in the world, and it is also the second largest staple food crop in my country. Therefore, ensuring the high quality and high yield of wheat is very important for ensuring the food security and quality of life of people in my country and the world. With the increase of planting density, changes in fertilizer and water conditions and farming systems, soil-borne fungal diseases such as wheat sheath blight, root rot, and stem-base rot have developed into major rhizome diseases in my country's wheat production, seriously affecting the quality of wheat grains. Yield and quality have become one of the main problems to be solved urgently in my country's wheat production. Wheat sheath blight, also known as wheat sharpeyespot. Wheat sheath...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8218C12N15/8282
Inventor 张增艳吴天赐郭飞龙
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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