Method for knocking out bnaFAH gene of brassica napus by using CRISPR-cas9 system and application

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a method for knocking out BnaFAH gene of Brassica napus by using a CRISPR-Cas9 system and application. Two copies of the BnaFAH gene of Brassica napus are knocked out by using a CRISPR-Cas9 system, two target fragments are selected from the fourth and sixth exon conservative regions of the two copies of the BnaFAH gene of Brassica napus, and BnaFAH-Cas9 plant expression vectors are constructed respectively, the vectors are introduced into hypocotyl callus of Brassica napus by a method of agrobacterium infection, seedlings are regenerated, double-strand cleavage is carried out on the two copies of the BnaFAH gene under the joint action of two sgRNAs and Cas9 nucleases, random deletion on the target gene fragment is finally realized by the self-repairing function of cells, gene knockout is completed, and mutant plants are obtained. It is verified that the mutant plants obtained after the BnaFAH gene is knocked out enhance the resistance to plutella xylostella, and it is indicated that the gene is a key gene for regulating the insect resistance of Brassica napus, and has important significance for breeding new Brassica napus varieties with insect resistance.

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