Method for site-directed mutation of rice CENH3 gene by using CRISPR-CAS9 technology

A gene site-directed mutation, CRI-R2 technology, applied in recombinant DNA technology, horticultural methods, genetic engineering, etc., can solve the problems of cytotoxicity, cumbersome process, high off-target rate, etc.

Inactive Publication Date: 2017-05-31
HUAZHI RICE BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional methods of creating mutants include physical mutagenesis, chemical mutagenesis, etc., which are blind and require a lot of screening work.
In the past few years, sequence-specific nuclease technologies represented by ZFN (zinc-finger nucleases) and TALEN (transcription activator-like effector nucleases) can achieve site-specific genome editing, but they have disadvantages such as cytotoxicity, cumbersome process, and high off-target rate.

Method used

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  • Method for site-directed mutation of rice CENH3 gene by using CRISPR-CAS9 technology
  • Method for site-directed mutation of rice CENH3 gene by using CRISPR-CAS9 technology
  • Method for site-directed mutation of rice CENH3 gene by using CRISPR-CAS9 technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Construction of carrier pHZ1-CAS9-gRNA-CENH3

[0073] 1. Selection of target sites in rice CENH3 (LOC_Os05g41080) gene

[0074] Log in to the plant transcription factor database (http: / / rice.plantbiology.msu.edu / analyses_search_locus.shtml), query the sequence of the rice CENH3 gene (LOC number: LOC_Os05g41080), and design the sgRNA sequence based on CRISPR / Cas9.

[0075] The nucleotide sequence of the sgRNA action site is 5'-GTCCAGGCACAGTGGCACTG-3'.

[0076] 2. Construction of the carrier pHZ1-CAS9-gRNA-CENH3

[0077] 1) Construction of OsU6-CENH3-gRNA-polyT fragment

[0078] In the first round of PCR, the Cris-GL3 plasmid was used as a template, and the primers CRI-F1 and SQ2-2-R1 were used to amplify the 20bp target sequence of the OsU6 promoter and the CENH3 gene, and the fragment size was 329bp; Primers SQ2-2-F2 and CRI-R2 amplify the 20bp target sequence of CENH3 gene and gRNA-polyT, the fragment size is 180bp; the second round of PCR uses the first r...

Embodiment 2

[0088] Example 2 Preparation method of rice CENH3 gene site-directed mutation plant

[0089] 1. Agrobacterium transformation and colony PCR identification

[0090] Take the competent Agrobacterium from the -80°C refrigerator and thaw it on ice, add 3-5 μL of plasmid pHZ1-CAS9-gRNA-CENH3 and mix well, put it on ice for 30 minutes, put it in liquid nitrogen for 1 minute, and then heat shock it in a 37°C water bath 1min, add 1mL of YEP medium to the aseptic operating platform, shake on a shaker at 28°C for 2-4h, take it out, centrifuge at 8000rpm / min for 1min, pour off part of the supernatant in the aseptic operating platform, and spread the remaining medium evenly with a spreader. The suspension below was mixed and spread on the YEP plate added with rifampicin and kanamycin, sealed with parafilm and placed in a 28°C incubator for cultivation. After 2 days, pick a single colony, shake the bacteria at 28°C for 24-36 hours, and perform colony PCR to obtain positive colonies.

[0...

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Abstract

The invention provides a method for site-directed mutation of rice CENH3 gene by using a CRISPR-CAS9 technology. A CRISPR-CAS9-based sgRNA sequence is designed for the rice CENH3 gene, and a DNA fragment containing the sgRNA sequence is connected to a vector carrying the CRISPR/Cas to convert rice in order to realize the site-directed mutation of rice CENH3 gene, wherein the nucleotide sequence of the sgRNA action site is represented by SEQ ID NO:1. Rice endogenous gene CENH3 is edited through the CRISPR-CAS9 technology to obtain a CENH3 mutant, so a CENH3 mediated chromosome elimination mechanism is hopeful to be applied to the double haploid breeding process of rice.

Description

technical field [0001] The invention relates to the field of plant transgenic technology and the field of crop genetic breeding, in particular to a method for site-directed mutation of rice CENH3 gene using CRISPR-CAS9 technology. Background technique [0002] In rice haploid breeding, at present, plant tissue culture, such as in vitro culture of anthers, is mainly used to induce haploid plants, and then restore the normal number of chromosomes to plants through chromosome doubling. The efficiency of haploid induction in tissue culture varies for various rice varieties and takes a long time. [0003] The centromere-specific histone CENH3 plays a key role in the assembly of centromere and the normal segregation and transmission of chromosomes. In Arabidopsis, the modified CENH3 gene was introduced into the cenh3 Arabidopsis mutant through transgenic technology, and this was used as the female parent to achieve chromosome elimination after crossing with the wild-type male par...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00
CPCC07K14/415C12N15/8205
Inventor 沈倩李继明徐冉
Owner HUAZHI RICE BIO TECH CO LTD
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