一个线虫长非编码RNA的单细胞精度表达谱分析以及基因敲除研究

Analysis of Expression Pattern in Single Cell Resolution of an lncRNA and Gene Knock-out in C. elegans

秀丽隐杆线虫(Caenorhabditis elegans,C.elegans)中长非编码RNA(long non-protein coding RNA gene,lncRNA)的结构和功能是近年来的研究热点之一。数据表明长非编码基因linc-5在线虫的胚胎和早期幼虫均有较高表达,提示其在线虫的胚胎发育过程中具有非常重要的作用。选取线虫两个物种C.elegans和C.briggsae为实验材料,构建启动子与荧光报告基因(mCherry)融合载体;通过基因枪整合到基因组上,得到纯合转基因线虫品系;结合在线虫中建立的单细胞精度的表达谱分析技术,对线虫两个物种幼虫L1时期的表达谱比较,说明linc-5在物种间具有非常强的保守性;在线虫中利用基因组编辑工具CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated system)技术成功敲除linc-5基因,为进一步研究linc-5的功能提供了良好基础。

Long non-protein coding gene （lncRNA） in C. elegans gains great mania in recent years, linc-5 is a lncRNA which has high expression level in embryo and early larva according to the RNA-seq data, suggesting its importance during the development of C. elegans. Based on two species C. elegans and C. briggsae, expression vector of promoter and fluorescent protein （mCherry） fusion was constructed as reporter gene. Reporter gene was integrated to genome using gene bombardment and got homozygous transgene strains of C. elegans and C. briggsae. Then the expression pattern was compared in L1 larva stage using the well-established technique of analysis in single cell resolution, and it showed strong conservation between the two species. Through the genome editing tools CRISPR/Cas9, linc-5 was knocked out successfully, which provided infrastructure for further study on lncRNA.