CRISPR/Cas9技术敲除水稻BR降解相关基因的研究

Rice (Oryza sativa) BR Degradation Related Genes Knockout by CRISPR/Cas9 Technology

油菜素内酯(BR)是一种常见的植物类激素,在植物的生长发育过程中起重要的调控作用,对水稻株型改良和抗逆性有重要作用。研究首先构建了水稻4个BR降解相关基因的CRISPR/Cas9载体;然后利用农杆菌介导法,将构建好的4个BR降解相关基因的CRISPR/Cas9载体分别转化水稻中花11的愈伤组织,用50 mg/L的潮霉素作为筛选标记,共获得395株转基因植株。其中,有270株属于野生型,61株属于杂合子类型,64株属于突变类型;64株突变体中有39株是杂合突变体,25株是纯合突变体,纯合突变的概率为6.25%。4个基因均获得了突变材料,但不同基因的突变效率不同,其中转LW26的植株突变率高达41%。

Brassinolide(BR)as a common plant hormone plays an important regulation role in the growth and development ofplants,especially in the rice plant type improvement and stress resistance.In this study,CRISPR/Cas9knockout carriers of four riceBR degradation related genes were constructed,then the carriers of the four genes were transformed into rice(Oryza sativa L.)viaAgrobacterium tumefaciens-mediated genetic transformation of rice variety Zhonghua11callus,and395transgenic plants were obtainedafter the callus was screened,each carrier with50mg/L hygromycin as a screening marker;and PCR product was amplified with theprimers on both sides of the target site and sequenced.The results showed that the395transgenic plants included270plants belongingto wild type,61to heterozygote type and64to mutant type.Of the64mutants,39were heterozygous mutants and25were homozygousmutants,and the frequency of homozygous mutation is6.25%.The mutant alleles were found in all the four genes,but the mutationefficiencies of different genes were different;and among them,the mutation frequency of the LW26transgenic plants was41%.Thesetransgenic plants could be used as a foundation for the breeding of homozygous progeny and the identification of mutation expression inthe future.