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Method for improving wheat disease resistance by inhibiting expression of TaVQ5 gene in wheat

A technology of disease resistance and genetics, applied in genetic engineering, plant genetic improvement, chemical instruments and methods, etc., can solve the problems of identification of wheat VQ members and biological functions that need to be further studied

Active Publication Date: 2020-11-20
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

Due to the large wheat genome and allohexaploidy, the identification and biological functions of wheat VQ members need further study

Method used

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  • Method for improving wheat disease resistance by inhibiting expression of TaVQ5 gene in wheat
  • Method for improving wheat disease resistance by inhibiting expression of TaVQ5 gene in wheat
  • Method for improving wheat disease resistance by inhibiting expression of TaVQ5 gene in wheat

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1, preparation recombinant plasmid

[0059] Artificially synthesized recombinant plasmid pCXUN-Cas9-gRNA. The recombinant plasmid pCXUN-Cas9-gRNA is a circular plasmid. The nucleotide sequence of the recombinant plasmid pCXUN-Cas9-gRNA is shown in Sequence 1 of the sequence listing. From the 5' end in sequence 1, the 115th-367th nucleotides are reverse complementary to the NOS terminator, the 392-4522th nucleotides are reverse-complementary to the coding gene of the Cas9 protein, and the 4544-6533rd nucleotides The acid is reverse complementary to the Ubi promoter, the 6552-6914th nucleotide is the U6 promoter, and the 6915-7017th nucleotide is the coding gene of the sgRNA (the 6915-6934th nucleotide is the target sequence recognition region ). The recombinant plasmid pCXUN-Cas9-gRNA expresses sgRNA, and the target sequence of the sgRNA is located in the wheat TaVQ5 gene, and the specific target sequence is shown in sequence 8 of the sequence listing.

[...

Embodiment 2

[0061] Example 2. Using sgRNA to obtain gene-edited wheat by gene gun method

[0062] 1. Genetic transformation of wheat mediated by gene gun

[0063] 1. The young embryos of wheat variety Zhengmai 7698 about 14 days after pollination were taken as explants. The explants were inoculated in hypertonic medium, and cultured in the dark at 22-25°C for 1-2 days.

[0064] Hyperosmotic medium: MS basic medium with sucrose added, the sucrose concentration is 180g / L.

[0065] 2. Use the recombinant plasmid pCXUN-Cas9-gRNA as the DNA to be transformed, and use the PDS-1000 / He gene gun of BIO-RAD Company to bombard the immature embryos that have completed step 1 (Psi900, 27.5cmHg column), and the immature embryos after bombardment Transfer to induction medium and culture in dark at 22-25°C for 2-3 weeks.

[0066] The induction medium is based on MS medium, supplemented with Vc (vitamin C), 2,4-D (2,4-dichlorophenoxyacetic acid) and sucrose, and the concentration of Vc in the induction...

Embodiment 3

[0114] Embodiment 3, the detection of character

[0115] 1. Material acquisition

[0116] Edited plants for testing: TaVQ5-34, TaVQ5-74, TaVQ5-255 obtained in Example 2 were self-crossed to obtain T 1 Generation plants, the T that will be selected 1 Generation plants were selfed to obtain T 2 Generation seed, according to the method for embodiment 2 to each T 2 Generation plants were identified from T 2 In the generation plants, the homozygous mutation type based on the target sequence was selected, and the plants that did not carry the vector sequence (TaVQ5-34-2-9, TaVQ5-74-2-11, TaVQ5-74-2-19, TaVQ5-74-6 T 3 Generation seed is T 3 Generation of plants that do not carry the vector sequence. will originate from T 0 T of generation plant TaVQ5-34 3 Plants that do not carry the vector sequence in the generation are named T 3 Generation TaVQ5-34, will originate from T 0 T of generation plant TaVQ5-74 3 Plants that do not carry the vector sequence in the generation ar...

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Abstract

The invention discloses a method for improving wheat disease resistance by inhibiting the expression of a TaVQ5 gene in wheat. A CRISPR / Cas9 technology is adopted to edit the TaVQ5 gene at a fixed point, and by causing frameshift mutations, the wheat TaVQ5 gene is knocked out, and a new wheat germplasm with significantly improved resistance to powdery mildew and sheath blight is obtained. The method improves the ability of wheat to resist powdery mildew and sheath blight, innovates wheat disease-resistant germplasm resources, has important significance for new variety cultivation, environmental sanitation and food safety, and has great application and promotion value.

Description

technical field [0001] The invention relates to a method for improving disease resistance of wheat by inhibiting the expression of TaVQ5 gene in the field of biotechnology. Background technique [0002] Wheat (Triticun aestivum L.), as one of the most important food crops in the world, provides basic protein and carbohydrates for humans, enhances the resistance to diseases and insect pests of wheat varieties, and improves yield and quality characteristics, which are important goals for wheat improvement. [0003] Wheat powdery mildew is an epidemic disease caused by wheat powdery mildew (Blumeria graminis f.sp.tritici), and it is one of the main diseases in wheat production. Wheat powdery mildew can occur and cause damage at the seedling stage and the adult plant stage. Wheat powdery mildew affects the aboveground organs of wheat plants, mainly the leaves. When the disease is severe, it can also damage the stems and ears. 5% to 34% yield loss. [0004] Wheat sheath blight ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/29C07K14/415A01H5/00A01H6/46
CPCC12N15/8218C12N15/8282C07K14/415
Inventor 夏兰琴田金福李少雅
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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