Application of OsDSK2a protein or encoding gene thereof in regulation and control of resistance of rice to rice blast

A rice blast, encoding gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, to achieve the effects of enhanced disease resistance, improved rice blast resistance, and reduced rice blast susceptibility

Pending Publication Date: 2021-02-09
AGRO BIOLOGICAL GENE RES CENT GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the identification, cloning and application of rice susceptibility genes

Method used

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  • Application of OsDSK2a protein or encoding gene thereof in regulation and control of resistance of rice to rice blast
  • Application of OsDSK2a protein or encoding gene thereof in regulation and control of resistance of rice to rice blast
  • Application of OsDSK2a protein or encoding gene thereof in regulation and control of resistance of rice to rice blast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Selection of Os10g0542200 Gene Knockout Target Site and Construction of Knockout Vector

[0024] 1. Selection of knockout target sites

[0025] A knockout target sequence was designed for the Os10g0542200 gene (its nucleotide sequence is as SEQ ID No: 1). A 20bp specific target sequence was screened using E-CRISP (http: / / www.e-crisp.org / E-CRISP / ) for generating sgRNA. The sequence (nucleotide sequence shown in SEQ ID NO.3) is located at 352-371bp of the second exon of Os10g0542200 gene, full-length 1728bp CDS sequence (nucleotide sequence shown in SEQ ID NO.1) ( 5'-GCTTCAGCTGCTCCTAGCAG-3'). The amino acid sequence of the encoded protein DSK2a of the Os10g0542200 gene is shown in SEQ ID NO.2.

[0026] 2. Construction of knockout vector pYLCRISPR / Cas9-OsDSK2a

[0027] (1) Strain activation and plasmid extraction and preparation: The plasmid containing pYLCRISPR / Cas9-H (AddgeneID66187) from the laboratory of Academician Liu Yaoguang of South China Agricultura...

Embodiment 2

[0037] Example 2 Obtaining and Identification of Os10g0542200 Gene Knockout Transgenic Plants

[0038] The constructed knockout vector pYLCRISPR / Cas9-OsDSK2a was transformed into the normal japonica rice variety Nipponbare by using the genetic transformation method mediated by Agrobacterium EHA105. The rice transformation work was entrusted to Wuhan Boyuan Biotechnology Co., Ltd. The general process of transformation is as follows: the pYLCRISPR / Cas9-OsDSK2a plasmid was extracted and transformed into Agrobacterium EHA105, and the Agrobacterium EHA105 containing the pYLCRISPR / Cas9-OsDSK2a plasmid was used to infect the callus of the japonica rice variety Nipponbare, and then transferred to the co-culture medium Culture in dark at 26°C for 2 to 4 days, and transfer the washed callus to the selection medium containing hygromycin for resistance selection, and transfer the selected resistant callus to the pre-differentiation medium for 10 to 14 days. Then transfer to the different...

Embodiment 3

[0040] Example 3 Os10g0542200 Gene Knockout Transgenic Plants Leaf Blast Phenotypic Identification of Rice Blast Resistance

[0041] The homozygous 3 gene knockout T0 generation lines (OsDSK2aCas9-6, OsDSK2aCas9-14, OsDSK2aCas9-17) and wild-type rice seeds germinated at 32°C in Example 2, and after 2 days, the young shoots were moved to the Seeds were sown in plastic trays of soil, and when the seedlings grew to the 3-4 leaf stage, they were transplanted into black plastic barrels (about 30 cm in diameter and about 45 cm in height), with 4 plants in each barrel, and 16 plants in each strain. Four weeks later, the method of punching inoculation was used to identify the resistance to leaf blast. The second or third leaf of each plant was punched with a hole punch, and then 10 microliters containing 5x10 5 The bacterium solution of a spore (Pyriculariaoryzae Cav.) was dripped onto the leaf wound, and then the leaves were stuck with waterproof tape to prevent the bacterium soluti...

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Abstract

The invention discloses application of an OsDSK2a protein or an encoding gene thereof in regulation and control of the resistance of rice to rice blast. The amino acid sequence of the OsDSK2a proteinis shown in SEQ ID NO. 2. The invention, for the first time, proves that the rice DSK2a gene (Os10g0542200) is a functional gene for rice blast infection, and cloning and biological function verification of the gene have important reference significance for studying molecular mechanisms of the resistance of rice to rice blast. The invention provides an Os10g0542200 gene editing vector mediated byCas9. After rice is transformed by the vector, the expression level of Os10g0542200 can be greatly reduced, the susceptibility of the transformed plant to the rice blast is reduced along with the reduction of the expression level, the disease resistance is obviously enhanced, and there is no obvious change in growth state and agronomic traits of a transgenic plant. According to the invention, theCas9-mediated Os10g0542200 gene knockout technology can be applied to genetic engineering breeding of rice, and can be applied to production practice to improve the resistance of rice to the rice blast, so that the rice production safety is guaranteed under the current climatic condition of frequently occured rice diseases.

Description

Technical field: [0001] The invention belongs to the technical field of crop genetics, and in particular relates to the application of OsDSK2a protein or its coding gene in regulating rice blast resistance. Background technique: [0002] Rice blast caused by the fungus Magnaporthe grisea (Hebert) Barr is one of the most serious rice diseases in the world, and has a devastating impact on global rice cultivation. Rice blast may occur in all stages of rice growth, especially leaf blast and ear blast are the most harmful. The global annual output loss due to rice blast is 11-30%, the direct economic loss is about 5 billion US dollars, and the lost grain is enough to feed 60 million people. In my country, rice blast occurs wherever rice is cultivated. The northern and southern rice fields are affected to varying degrees every year, generally with a 10-20% reduction in production, and 40-50% in severe cases, and some fields even have no harvest. [0003] At present, using the r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8282
Inventor 晏石娟刘清李文燕陈中健孔谦黄文洁
Owner AGRO BIOLOGICAL GENE RES CENT GUANGDONG ACADEMY OF AGRI SCI
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