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Method for site-directed mutation of maize gene by means of CRISPR/Cas9 system

A gene site-directed mutagenesis, cas9-gfp-gRNA technology, applied in genetic engineering, botany equipment and methods, biochemical equipment and methods, etc.

Active Publication Date: 2017-08-08
先正达集团股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Maize is the most important food and feed crop in the world, and there is no report on the application of CRISPR / Cas system in maize breeding

Method used

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  • Method for site-directed mutation of maize gene by means of CRISPR/Cas9 system
  • Method for site-directed mutation of maize gene by means of CRISPR/Cas9 system
  • Method for site-directed mutation of maize gene by means of CRISPR/Cas9 system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction of a CRISPR / Cas9 system targeting the GFP gene in the Maize Xiang 249 inbred line

[0038] 1. Design the sgRNA sequence based on CRISPR / Cas9 for the GFP gene in the maize Xiang 249 inbred line. Wherein, the nucleotide sequence of the sgRNA action site is 5'-ACCGGGGTGGTGCCCATCC-3'.

[0039] 2. Ligate the DNA fragment containing the sequence encoding the sgRNA into a vector carrying CRISPR / Cas to construct the vector Cas9-GFP-gRNA for transforming maize. Wherein, the full sequence of the carrier Cas9-GFP-gRNA is shown in SEQ ID NO:2.

Embodiment 2

[0040] Example 2 Preparation method of gene site-directed mutation maize plant

[0041] 1. Treatment of corn ears and separation of immature embryos

[0042] (1) Maize Xiang 249 inbred line T1 generation transgenic maize plants carrying exogenous GFP gene were planted in the greenhouse. About 10 days after pollination, when the immature embryos grew to 0.8-2.0 mm, corn cobs were harvested, bracts were removed, and prepared sterilization;

[0043] (2) Dilute the sodium hypochlorite mother solution with a concentration of 6-15% to 15%-20% by volume with sterilized water, add Tween-20% by 1 drop (20μL) / 3L and mix to make a sterilized solution;

[0044] (3) Put the cobs of corn into the sterilizing solution and soak for 15 minutes, rinse with sterile water 3-5 times for later use;

[0045] (4) Peel off the top of the seeds with a sterile scalpel blade, dig out the endosperm with a sterile spatula to expose the immature embryos from the seeds, and strip the immature embryos. Pla...

Embodiment 3

[0067] Example 3 Detection of gene site-directed mutation maize

[0068] 1. Observation on the expression of GFP gene in site-directed mutation maize tissue

[0069] Take the resistant callus after site-directed mutation of the GFP gene in Example 2, observe the expression of the GFP gene in the callus, and find that green fluorescence is seen under ultraviolet light, indicating that the GFP gene in this part of the callus has not been targeted Mutation, if there is no green fluorescence under ultraviolet light, it means that the GFP gene has been inactivated, and the results are shown in figure 1 . From figure 1 It can be seen that the two newly born glyphosate-resistant calli have no expression of GFP, but the non-resistant callus has GFP expression ( figure 1 B, D). It shows that the GFP gene is normally expressed in the untransformed cells, but the GFP gene is no longer expressed in the transformed cells, indicating that the GFP gene has been successfully edited and in...

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Abstract

The invention provides a method for site-directed mutation of a maize gene by means of a CRISPR / Cas9 system. An sgRNA sequence based on CRISPR / Cas9 is designed aiming at a target gene in maize, a DNA fragment for encoding the sgRNA sequence is connected to a vector carrying CRISPR / Cas9, and then maize transformation is conducted, so that site-directed mutation of a specific gene in maize is realized. Further, the vector containing CRISPR / Cas9 is transferred to an acceptor material carrying the target gene by means of a genetic transformation method, so as to obtain a site-directed mutation regeneration plant of the target gene. By the adoption of the method, site-directed mutation of a maize genome can be achieved. The method has the advantages that the experimental period is short, and operation is easy. Different target genes can be transformed in a site-directed mode by means of CRISPR / Cas9 systems with different targeting directions, a novel method is provided for improved breeding of maize, and the method is of great practical significance for maize trait improvement.

Description

technical field [0001] The invention relates to the field of plant transgenic technology and the field of crop genetics and breeding, in particular to a method for site-directed mutation of maize genes by using the CRISPR / Cas9 system. Background technique [0002] With the continuous development of biotechnology, more and more new breeding techniques are emerging. The traditional breeding technology has a long cycle, and it is difficult to make fixed-point and directional improvement on the target shape. The biotechnology breeding that has emerged in recent years has greatly shortened the breeding cycle, and can purposefully introduce new traits or improve inherent traits. [0003] CRISPR (clustered regularly interspersed short palindromic repeats) / Cas is an acquired immune system derived from bacteria and archaea. It uses target site-specific RNA to guide the Cas protein to modify the target site sequence, thereby forming various types of mutations in the target sequence....

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00
CPCC07K14/43595C12N15/8205C12N15/8216C12N2800/80C12N2810/10
Inventor 胡燕琳杨桥王文舒许洁婷唐通黄磊旷乐左丹汤益周倩周正剑刘涛章旺根马崇烈成雄鹰
Owner 先正达集团股份有限公司
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