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Editing modification method of piricularia oryzae resistance locus DNA in rice genome and editing vector

A gene editing and genome technology, applied in the field of genetic engineering, can solve the problems of obtaining disease-resistant varieties and long breeding cycle

Active Publication Date: 2019-11-08
SAAS BIOTECH & NUCLEAR TECH RES INST +1
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  • 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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  • Editing modification method of piricularia oryzae resistance locus DNA in rice genome and editing vector
  • Editing modification method of piricularia oryzae resistance locus DNA in rice genome and editing vector
  • Editing modification method of piricularia oryzae resistance locus DNA in rice genome and editing vector

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] One, construct a kind of vector plasmid PMF115 that contains geminivirus replication-related originals and is used for cloning fragment RHR for recombinant DNA, such as figure 2 shown.

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

[0098] 1. First, design and synthesize the small space region SIR (Seq No.8) and the large space region LIR (Seq No.9) according to the genome sequence of the wheat dwarf virus (Wheat dwarf virus) that infects wheat (GenBank accession number: DQ868525.1). ) and three DNA fragments of replication-associated protein Rep / RepA (Seq No.10).

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

[0100] The large interregional region (LIR) DNA fragment was synthesized by BGI and cloned on the plasmid pMV;

[0101] The DNA fragment of the replication-related protein Rep / RepA, the BsaI site contained in the original gene DNA sequence has bee...

Embodiment 2

[0126] There is a rice blast resistance gene locus on chromosome 6 of the rice genome, and 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 the rice blast resistance in the rice genome .

[0127] In this example, the vector of the present invention is used to recombine the artificially designed DNA fragments into the locus, so as to achieve the purpose of modifying and improving the disease-resistant genes. The specific operation steps are as follows:

[0128] 1. Construction of editing vector

[0129] In the first step, the target sequence of the sgRNA is determined according to the DNA sequence (Seq No.1) of a rice blast resistance locus in the rice genome:

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

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

[0132] In the second step, construct two sgRNA expression cassettes based on the ident...

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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 in particular relates to a method for editing and modifying rice blast resistance site DNA in a rice genome, and an editing carrier. Background technique [0002] The rice blast fungus (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 of the world and poses a serious threat to rice production. To deal with the epidemic of rice blast, breeding disease-resistant rice varieties 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 output rice. [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 ...

Claims

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Application Information

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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