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Rice receptor cytoplasm kinase OsRLCK22 and coding gene and application thereof

A technique for encoding genes and transgenic plants, applied in the field of rice receptor cytoplasmic kinase OsRLCK22 and its encoding genes and applications, which can solve the problems of narrow resistance spectrum and difficulty in utilization

Active Publication Date: 2020-02-21
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

Breeding disease-resistant varieties with resistance genes is currently the most economical and effective measure to control rice bacterial blight. However, 42 rice bacterial blight resistance genes / loci have been reported so far ( http: / / www.shigen.nig.ac.jp / rice / oryzabase / gene / list ) most of them show a narrow resistance spectrum or are difficult to use, and only genes such as Xa3, Xa4, Xa21 and Xa23 are widely used in production

Method used

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  • Rice receptor cytoplasm kinase OsRLCK22 and coding gene and application thereof
  • Rice receptor cytoplasm kinase OsRLCK22 and coding gene and application thereof
  • Rice receptor cytoplasm kinase OsRLCK22 and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1. Site-directed knockout of rice OsRLCK22 gene based on Fn-CRISPR / Cpf1 system

[0085] 1. Sequence and analysis of rice receptor cytoplasmic kinase gene OsRLCK22

[0086] Shown in the sequence SEQ ID No.2 of rice receptor cytoplasmic kinase gene OsRLCK22, sequence analysis shows that this gene comprises 5 exons altogether, respectively the 1st-351st of SEQ ID No.2 sequence (the first exon exon), 458-851 (the second exon), 1338-1469 (the third exon), 1927-1968 (the fourth exon), 2276-3237 (the third exon) five exons).

[0087] In the present invention, the sequence on the fifth exon of the rice receptor cytoplasmic kinase gene OsRLCK22 is used as the target sequence of the rice receptor cytoplasmic kinase gene OsRLCK22 knockout method based on CRISPR / Cpf1 technology.

[0088] 2. Primer design of Fn-CRISPR / Cpf1 vector and construction of recombinant expression vector

[0089] 2.1 Selection of Fn-CRISPR / Cpf1 Technology Target Sequence

[0090] The Fn-CRISPR / Cp...

Embodiment 2

[0099] Example 2. Application of Fn-CRISPR / Cpf1 technology-based knockout method in rice varieties

[0100] Recombinant Agrobacterium EH105-Fn-OsRLCK22-1 was used to infect callus induced by mature embryos of rice variety Nipponbare, and the obtained rice positively transformed plants were named NIP-Fn-OsRLCK22-1.

[0101] The specific method is as follows:

[0102] 1. Inoculate the recombinant Agrobacterium EH105-Fn-OsRLCK22-1 in YEB liquid medium (containing 50 μg / ml kanamycin and 20 μg / ml rifampicin), and culture with shaking at 28°C and 200 rpm until the OD600 is 0.6 -0.8; Centrifuge at 5000rpm, 4°C for 5min, resuspend the bacterial cell pellet concentration in AAM liquid medium (acetosyringone concentration is 200μM / L, pH5.2) to OD600 of 0.6-0.8, and obtain recombinant Agrobacterium suspension .

[0103] 2. Remove chaff from mature seeds of rice variety Nipponbare, soak in 75% ethanol for 1 min, then sterilize in NaClO solution (mix with water 1:2, add 1 drop of Tween 2...

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Abstract

The invention discloses a rice receptor cytoplasm kinase OsRLCK22 and a coding gene and application thereof. The invention firstly discloses an application of any one of the following proteins in regulating and controlling the bacterial blight resistance of plants: A1) a protein of which the amino acid sequence is SEQ ID No. 1; A2) a fusion protein obtained by connecting a label to N terminal or / and C terminal of the amino acid sequence shown as the SEQ ID No.1; and A3) a protein which is obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in the SEQ ID No.1 and has more than 90% of identity and same functions as the protein shown in the A1). The invention further discloses a method for cultivating a transgenic plant withenhanced bacterial blight resistance. The invention provides an efficient breeding mode for creating a bacterial blight resistant material based on the receptor cytoplasm kinase OsRLCK22, and the modehas important application value in agricultural production.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, it relates to rice receptor cytoplasmic kinase OsRLCK22 and its coding gene and application. Background technique [0002] Bacterial blight caused by Xanthomonas oryze pv. % or so, seriously up to 50%. Breeding disease-resistant varieties with resistance genes is currently the most economical and effective measure to control rice bacterial blight. However, 42 rice bacterial blight resistance genes / loci have been reported so far ( http: / / www.shigen.nig.ac.jp / rice / oryzabase / gene / list ) most of them showed a narrow resistance spectrum or were difficult to use, and only genes such as Xa3, Xa4, Xa21 and Xa23 were widely used in production. [0003] There is a specific interaction between rice and X.oryzae pv.oryzae. X.oryzae pv.oryzae is easy to mutate, and the co-evolution of rice X.oryzae pv.oryzae leads to the loss of variety resistance. Identifying and knocking out bacterial bligh...

Claims

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Application Information

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IPC IPC(8): C12N9/12C07K19/00C12N15/54C12N15/82A01H5/00A01H6/46
CPCC07K2319/21C07K2319/23C07K2319/24C07K2319/41C07K2319/42C07K2319/43C12N9/12C12N15/8218C12N15/8281
Inventor 周永力李全林史晓荣
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI