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A method for editing and modifying rice blast resistance locus dna in rice genome, and editing vector

A modification method and genome technology, applied in the field of genetic engineering, can solve the problems of long breeding cycle and obtaining disease-resistant varieties, etc.

Active Publication Date: 2020-08-11
SAAS BIOTECH & NUCLEAR TECH RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The conventional method of improving the resistance of rice varieties to rice blast is to adopt the traditional method of cross breeding. However, in the process of cross breeding, the cultivation of rice blast resistant varieties not only takes a long time, but it is often difficult to obtain corresponding disease-resistant varieties according to the set breeding goals.

Method used

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  • A method for editing and modifying rice blast resistance locus dna in rice genome, and editing vector
  • A method for editing and modifying rice blast resistance locus dna in rice genome, and editing vector
  • A method for editing and modifying rice blast resistance locus dna in rice genome, and editing vector

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Experimental program
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Effect test

Embodiment 1

[0096] 1. Construct a vector plasmid PMF115 that contains the original copy of the geminivirus and is used to clone the RHR fragment for recombinant DNA, such as figure 2 Shown.

[0097] The construction method of plasmid PMF115 is as follows:

[0098] 1. First, according to the wheat dwarf virus (Wheat dwarf virus) genome sequence (GenBank accession number: DQ868525.1) designed and synthesized the small area SIR (Seq No. 8), the large area LIR (Seq No. 9) ) And replication-related protein Rep / RepA (Seq No. 10) three DNA fragments.

[0099] The SIR DNA fragment was synthesized by BGI Gene (HTTP: / / WWW.GENOMICS.CN / ) and cloned on the plasmid PJWR;

[0100] The LIR DNA fragment was synthesized by BGI gene and cloned on the plasmid pMV;

[0101] The DNA fragment of replication-related protein Rep / RepA, the BsaI site contained in the original gene DNA sequence has been eliminated. This fragment was synthesized by BGI Gene and cloned on plasmid pMV.

[0102] 2. After the above-mentioned pla...

Embodiment 2

[0126] There is a rice blast resistance gene locus on the 6th chromosome of the rice genome. The variation of this locus produces different resistance to many physiological races of rice blast in the rice genome, and plays an important role in rice blast resistance. .

[0127] In this example, through the vector of the present invention, artificially designed DNA fragments were recombined into sites to achieve the purpose of modifying and improving disease-resistant genes. The specific steps are as follows:

[0128] 1. Construction of editing vector

[0129] The first step is to determine the target sequence of sgRNA based on the DNA sequence (Seq No.1) of a rice blast resistance site in the rice genome:

[0130] sgRNA-1 (forward): 5’-TTGAGTAGCAAACTAAAGGAAGG-3’;

[0131] sgRNA-2 (reverse complement): 5’-CCTCCCCTACTAAGGACACTCAG-3’;

[0132] The second step is to construct two sgRNA expression cassettes based on the determined target sites:

[0133] ①sgRNA-1 expression cassette

[0134] Sy...

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Abstract

The invention discloses a method for editing modification of DNA of a piricularia oryzae resistance locus in a rice genome by using a CRISPR / Cas9 gene editing vector. The piricularia oryzae resistancelocus in the rice genome is located on a chromosome 6, and the DNA sequence is shown as Seq No.1. The CRISPR / Cas9 gene editing vector implementing the editing modification method comprises an sgRNA expression cassette and a recombinant DNA fragment RHR; the target sequence of the sgRNA expression cassette is determined according to the DNA sequence of the piricularia oryzae resistance locus in the rice genome; and the recombinant DNA fragment RHR is constructed by a method combining DNA synthesis and PCR amplification according to the DNA sequence of a piricularia oryzae resistance gene. According to the method, the DNA of the piricularia oryzae resistance gene locus is accurately modified and edited, so that the purpose of changing the genetic information is achieved, and the method is used for cultivating rice varieties resistant to piricularia oryzae.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a method for editing and modifying the DNA of rice blast resistance sites in the rice genome and an editing vector. Background technique [0002] Magnaporthe oryzae infects rice and causes rice blast. Rice blast is one of the most devastating diseases in rice production. It is widely distributed in rice producing areas in the world and poses a serious threat to rice production. To deal with the epidemic of rice blast, breeding rice varieties with resistance to disease is the most economical and effective method. Compared with pesticide control, it can also greatly reduce the pollution of the ecological environment and effectively reduce the pesticide pollution in the rice output. [0003] The conventional method to improve the resistance of rice varieties to blast disease is to adopt the traditional method of cross breeding. However, in the process of cross breed...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/90C12N15/66A01H5/00A01H6/46
CPCC07K14/415C12N15/8282C12N15/902C12N2800/80C12N2810/10
Inventor 陈克贵彭梅芳潘春梅李邵怡王静范晓丽
Owner SAAS BIOTECH & NUCLEAR TECH RES INST