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Cultivating method for disease-resistant TaMYB-KW gene-transferred wheat, related biomaterials and application

A biomaterial and transgenic plant technology, applied in the cultivation of disease-resistant transgenic TaMYB-KW wheat and related biomaterials and application fields, can solve the problems of lack of wheat disease-resistant germplasm resources and slow research progress.

Inactive Publication Date: 2016-05-18
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because wheat sheath blight resistance is controlled by multiple genes, there is a lack of ideal wheat disease-resistant germplasm resources, and conventional breeding methods have made slow progress in the selection of wheat varieties resistant to sheath blight

Method used

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  • Cultivating method for disease-resistant TaMYB-KW gene-transferred wheat, related biomaterials and application
  • Cultivating method for disease-resistant TaMYB-KW gene-transferred wheat, related biomaterials and application
  • Cultivating method for disease-resistant TaMYB-KW gene-transferred wheat, related biomaterials and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Embodiment 1, the cloning of wheat disease resistance protein TaMYB-KW and its coding gene

[0091] The inventor of the present application isolated and cloned a wheat disease resistance protein from sheath blight resistant wheat CI12633, and named it TaMYB-KW. The specific cloning method of TaMYB-KW gene is as follows:

[0092] Take the leaves of wheat CI12633 seedlings inoculated with Rhizoctonia solani, treat them with liquid nitrogen, and extract the total RNA of the leaves according to the instructions of the Invitrogen TRIZOL Reagent total RNA extraction reagent. According to the procedures of Invitrogen's first-strand cDNA synthesis kit, the extracted RNA samples were reverse-transcribed to synthesize first-strand cDNA, which was used as a template for gene cloning.

[0093] In order to obtain the full-length cDNA sequence of TaMYB-KW gene, two pairs of primers were used to amplify by RACE and nested PCR. Using the primer pair TaMYB-KWA-U1 (5'-GCAGCATTTACCTTCGG...

Embodiment 2

[0094] Embodiment 2, TaMYB-KW gene is subjected to the induced expression analysis of wheat sheath blight pathogen

[0095] In order to study whether the expression level of TaMYB-KW gene is related to the resistance of wheat sheath blight, Q-RT-PCR was used to analyze the expression of TaMYB-KW gene in the sheath blight resistant wheat CI12633 inoculated with Rhizoctonia graminearum for 4 days and 10 days. Express the situation.

[0096] Inoculate between the leaf sheaths and stems of wheat CI12633 tillering stage seedlings with wheat sheath blight pathogenic bacteria-Rhizoctonia graminearum (Rhizoctoniacerealis) R0301 mycelia toothpicks and wheat grains; take wheat stems 4 days and 21 days after inoculation, liquid nitrogen Store in -80°C ultra-low temperature freezer after quick freezing.

[0097] Total RNA (about 5 μg total RNA per sample) was extracted from each wheat stem, and reverse-transcribed into cDNA according to the procedure of Invitrogen's first-strand cDNA syn...

Embodiment 3

[0103] Example 3, Acquisition of TaMYB-KW Silenced Wheat and Identification of Disease Resistance

[0104] 1. Construction of recombinant expression vector

[0105] 1. Inoculate wheat CI12633 stems and leaf sheaths with Rhizoctonia graminearum (Rhizoctoniacerealis) R0301, extract RNA after 4 days, reverse transcribe into cDNA; use cDNA as template, use TaMYB-KW-VIGS-F and TaMYB-KW-VIGS- The primer pair composed of R was subjected to PCR amplification to obtain a PCR amplification product (a TaMYB-KW fragment carrying an NheI site).

[0106] TaMYB-KW-VIGS-F:5'-ACA GCTAGC TCGTCCGCCACCGATTACT-3' (underlined as NheI enzyme recognition site);

[0107] TaMYB-KW-VIGS-R:5'-GGC GCTAGC CTCTGCCTAAATCTGAGACAAAC-3' (underlined as NheI enzyme recognition site).

[0108] PCR reaction program: first 94°C pre-denaturation for 3 minutes; then 94°C for 30s, 56°C for 30s, 72°C for 1min, 15 cycles; 94°C for 30s, 60°C for 30s, 72°C for 1min, 20 cycles; finally, 72°C for 10 minutes end.

[0...

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Abstract

The invention discloses a cultivating method for disease-resistant TaMYB-KW gene-transferred wheat, related biomaterials and application. TaMYB-KW protein is one of the proteins as follows: the first protein has an amino acid sequence shown in the sequence 2, the second protein is obtained in the mode that the amino acid sequence shown in the sequence 2 in a sequence table is subjected to substitution and / or deletion and / or addition of one or several amino acid residues and is related to the disease resistance of plants, and the third fusion protein is obtained by connecting labels to the N end or / and the C end of the first protein or the second protein. Experiments prove that the TaMYB-KW and a coding gene thereof can improve the disease resistance of the plants, and the TaMYB-KW gene is a wheat disease-resistant protein gene which is closely related to the sharp eyespot resistance and can be applied to genetic improvement of the plants.

Description

technical field [0001] The invention relates to a breeding method for disease-resistant transgenic TaMYB-KW gene wheat in the field of biotechnology, as well as related biological materials and applications. Background technique [0002] Wheat (Triticuma aestivum) is one of the four major crops that humans rely on for survival. More than one-third of the world's population uses wheat as a staple food. The yield and quality of wheat directly affect human survival and quality of life. With the changes of farming systems, fertilizer and water conditions, and climate conditions, soil-borne diseases such as sheath blight and root rot are increasing in my country's wheat production areas, and have become one of the important factors restricting high and stable yields of wheat. [0003] Wheat sheath blight, also known as wheat sharp eye spot (wheatsharpeyespot), is a worldwide soil-borne fungal disease of wheat, mainly caused by the saprotrophic trophic fungus Rhizoctonia gramina (...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N5/10A01N47/44A01P3/00A01H5/00
CPCA01N47/44C07K14/415C12N15/8282
Inventor 张增艳单天雷荣玮魏学宁杜丽璞
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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