Method for performing site-directed mutation on rice TDR gene by CRISPR\Cas9 system and detection method

A site-directed mutagenesis and detection method technology, applied in the field of high-resolution melting curve analysis, can solve the problems of difficult purification of genetic background, time-consuming and labor-intensive recessive male sterility gene tdr, etc.

Inactive Publication Date: 2020-08-25
江西省超级水稻研究发展中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the shortcomings of time-consuming and labor-intensive use of the ready-made recessive male sterility gene tdr in rice for backcrossing, and the shortcomings of difficult purification of the genetic background, the pre

Method used

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  • Method for performing site-directed mutation on rice TDR gene by CRISPR\Cas9 system and detection method
  • Method for performing site-directed mutation on rice TDR gene by CRISPR\Cas9 system and detection method
  • Method for performing site-directed mutation on rice TDR gene by CRISPR\Cas9 system and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1. Creating Recessive Male Sterile Lines in Rice by Knockout of TDR Gene by CRISPR\Cas9

[0063] Complete the knockout of the TDR gene (the gene number of the MSU rice genome database is LOC_Os02g02820; the number of the RAP rice genome database is Os02g0120500; its protein sequence is shown in SEQ ID No.1) and obtain the male sterile plant by the following steps:

[0064] 1) Design CRISPR\Cas9 system sgRNA guide sequence

[0065] Using the online tool CRISPR-P ( http: / / cbi.hzau.edu.cn / crispr / ), according to the method provided by the developer, design the guide sequence of the CRISPR\Cas9 system for the TDR gene (also equivalent to the target site sequence). This embodiment selects two guide sequences (that is, selects two target sites) for implementation. They are AGCACAGGTCTCAGCACTGC (ie, the sequence shown in SEQ ID No. 2, corresponding to target site 1) and GCTGCATTGAGGCCTCTAGT (ie, the sequence shown in SEQ ID No. 3, corresponding to target site 2).

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Embodiment 2

[0113] Example 2. Using high-resolution melting curve analysis to detect and propagate TDR mutation types that are easy to type with wild type

[0114]In Example 1, after obtaining sterile strains (homozygous mutant strains) of different gene-edited strains, the high-resolution melting curve method provided by the present invention is used to analyze the sterile strains of different strains together with the wild type to screen Mutant genotypes that are easy to type with wild type. The results showed that an easy-to-type mutation type was obtained from the mutant lines of target site 1 and target site 2 respectively, and the mutation numbers were #04 and ##07, respectively. Further genotyping detection was carried out on the fertile strains in the easy-to-type mutant lines, and heterozygous mutants were screened out (results of high-resolution melting curve analysis, see image 3 C and D), and then hybridize the heterozygous mutant fertile plants with the same line of sterile...

Embodiment 3

[0121] Example 3. Preliminary detection of TDR site-directed mutation T by high-resolution melting curve analysis 0 Gene mutations at the target sites of the subgenerational transformants

[0122] The TDR gene obtained in Example 1 was knocked out by the same method as described in Example 2. 0 The transformed seedlings were detected, and compared with the sequencing results, it was found that the high-resolution melting curves of all the plants with gene mutations were significantly different from those of the wild type (see image 3 A&B), therefore, using the method provided by the present invention to detect the site-directed mutation contemporary transformed plantlets can quickly determine whether there is a gene mutation at the target site of the transformant.

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Abstract

Recessive male sterility has an important utilization value in plant breeding. TDR gene function deficits in rice can cause thorough recessive male sterility, and is thus an important target of creating the recessive male sterility in genetic engineering. The invention discloses a method for performing site-directed mutation on a rice TDR gene by a CRISPR\Cas9 system. By using the site-directed mutation method provided by the invention, a TDR gene mutant can be fast and efficiently created so as to obtain a recessive male sterility material. The invention further provides a detection method ona specific TDR mutation genotype obtained by the site-directed mutation method. The detection method can be used for fast identifying whether the mutation exists in the target in a current generationtransformant of the TDR site-directed mutation, and can also be used for genotyping identification of the later-generation specific mutation genotype.

Description

technical field [0001] The invention belongs to the technical field of rice molecular breeding, relates to a method for performing site-directed mutation on rice TDR gene by using CRISPR\Cas9 system, and further provides a high-performance method for detecting the specific TDR mutation genotype obtained by the site-directed mutation method Resolution melting curve analysis method. Background technique [0002] Recessive male sterile lines have important application value in rice breeding, and have been successfully applied in recurrent selection breeding, and also in SPT technology (Seed production technology, a plant male sterile line production technology). Many recessive male sterility genes have been cloned in rice, among which the abortion caused by the inactivation of the TDR gene is very thorough, and the pollen-free abortion is easy to observe and identify in the field, and does not affect the agronomic traits other than fertility, so it has important application val...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H5/00A01H6/46C12Q1/6858
CPCC12N15/8205C12N15/8213C12N15/8289
Inventor 龙起樟黄永兰万建林王会民芦明唐秀英朱雪晶
Owner 江西省超级水稻研究发展中心
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