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Method for simultaneously improving rice fragrance and bacterial leaf blight resistance by using a CRISPR/Cas9 system and expression vector

A bacterial blight resistance and bacterial blight technology, applied in chemical instruments and methods, botany equipment and methods, biochemical equipment and methods, etc., can solve problems such as bad linkage, cumbersomeness, and prolonging the breeding cycle. Achieve efficient editing, improve breeding efficiency, and avoid biosafety evaluation problems

Active Publication Date: 2020-07-14
SHANGHAI AGROBIOLOGICAL GENE CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the aggregation of multiple genes is difficult to operate, and there is a linkage burden of bad traits in the process of creating the introduction line. It is necessary to backcross multiple generations or expand the size of the segregation population to break this linkage and prolong the breeding period. cycle

Method used

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  • Method for simultaneously improving rice fragrance and bacterial leaf blight resistance by using a CRISPR/Cas9 system and expression vector
  • Method for simultaneously improving rice fragrance and bacterial leaf blight resistance by using a CRISPR/Cas9 system and expression vector
  • Method for simultaneously improving rice fragrance and bacterial leaf blight resistance by using a CRISPR/Cas9 system and expression vector

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Experimental program
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Embodiment 1

[0057] Creating fragrance-resistant rice with bacterial blight resistance using CRISPR / Cas9 system

[0058] 1. Construction of double gene editing vector

[0059] 1.1 Target site selection and adapter primer design:

[0060] According to the genome sequence of aroma gene Badh2 (LOC_Os08g32870) and rice bacterial blight susceptibility gene SWEET14 (LOC_Os11g31190) promoter-specific binding elements, the target sequence was designed, and the 20nt nucleotide sgRNA target sequence was 5'-N20- NGG-3' sequence was designed.

[0061] The target site of aroma gene Badh2 is selected in the first exon region ( figure 1 A in A, a schematic diagram of the target site sequence of the aroma gene Badh2), its nucleotide sequence is:

[0062] Adapter Forward Primer 32870-Gf:

[0063] 5'-AGTGGCGCGCCCCCGCGCTgttttagagctagaaat-3' (SEQ ID NO.3)

[0064] Adapter reverse primer 32870-U6Ar:

[0065] 5'-AGCGCGGGGGCGCGCCACTCggcagccaagccagca-3' (SEQ ID NO.4)

[0066] The target site of the promote...

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Abstract

The invention relates to a method for simultaneously improving rice fragrance and bacterial leaf blight resistance by using a CRISPR / Cas9 system and an expression vector, and belongs to the technicalfield of rice improvement. The method utilizes the CRISPR / Cas9 system to inhibit the expression of a rice fragrance gene Badh2 and a bacterial leaf blight susceptible gene SWEET14; and in the CRISPR / Cas9 system, the nucleotide sequence of a guide RNA target site of the rice fragrance gene Badh2 is shown as SEQ ID NO. 1, and the nucleotide sequence of a guide RNA target site of the bacterial leaf blight susceptible gene SWEET14 is shown as SEQ ID NO. 2. The method can separate and obtain a mutant which has no transgenic marker, has fragrance and significantly improves the bacterial leaf blightresistance in the offspring, and provides a new germplasm resource and a creation method for breeding high-quality disease-resistant rice.

Description

technical field [0001] The invention relates to the technical field of rice improvement, in particular to a method and an expression vector for simultaneously improving rice aroma and bacterial blight resistance by using a CRISPR / Cas9 system. Background technique [0002] Rice (Oryzasativa L.) is the main food crop for about half of the world's population. With the improvement of people's living standards, people's requirements for rice quality are getting higher and higher. The aroma of rice is an important indicator of food quality. Fragrant rice has a special aroma and is widely favored by the market and consumers. [0003] Rice bacterial blight caused by the gram-negative bacterium Xanthomonas pv oryzae is one of the most serious bacterial diseases in rice production in the world, and can cause up to 50% yield loss in severe cases. [0004] In the process of conventional cross-breeding, there are few single plants of the homozygous genotype of fragrance (recessive trait...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29A01H5/00A01H6/46
CPCC12N15/8218C12N15/8281C12N15/8261C07K14/415
Inventor 刘毅张安宁刘国兰孔德艳徐凯王飞名余新桥罗利军
Owner SHANGHAI AGROBIOLOGICAL GENE CENT