大豆生物钟基因GmLNK1/2、GmRVE4/8和GmTOC1 CRISPR/Cas9组织表达分析及敲除靶点的鉴定

Identification of CRISPR/Cas9 knockout targets and tissue expression analysis of circadian clock genes GmLNK1/2,GmRVE4/8,and GmTOC1 in soybean

生物钟基因能够参与调控植物的整个生命进程,对提高作物产量具有重要的作用。LNK1(NIGHTLIGHTINDUCIBLE AND CLOCK-REGULATED 1)、LNK2、RVE4(REVEILLE 4)、RVE8(REVEILLE 8)和TOC1(TIMING OF CAB EXPRESSION1)是植物中重要的生物钟基因。本研究利用BLAST同源比对和进化树分析的方法分别鉴定AtLNK1、AtLNK2、AtRVE4、AtRVE8和AtTOC1在大豆中的同源基因,通过qRT-PCR实验证明这些生物钟基因在大豆根、茎、叶等组织中均有表达。成功构建这些基因的CRISPR/Cas9敲除载体,并利用大豆根毛转化体系成功鉴定出13个基因的CRISPR/CAS9有效靶点。为进一步获得稳定的大豆突变体材料及研究其生物钟基因的功能提供了理论基础。

Circadian clock genes play an important role in improving crop yield.LNK1,LNK2,RVE4,RVE8,and TOC1 are important circadian clock genes in plants.Homologous genes of At LNK1,At LNK2,AtRVE4,AtRVE8,and AtTOC1 in soybean were found by evolutionary tree analysis.These genes were expressed in soybean roots,stems and leaves.The knockout vectors of these genes were successfully constructed by using CRISPR/Cas9 gene editing technology.Transformation system of soybean root hair and RT-PCR were used to identify 13 genes targets effectively.The results of this study provide important target informations for further obtaining soybean mutant materials,and a foundation for further studying the function of circadian clock genes.