Sqt-1的5'端侧翼序列在旋毛虫rol6基因中的启动功能验证

Verification of 5' flanking sequence of Caenorhabditis elegans sqt-1 gene to promoter the expression of Trichinella spiralis rol6 gene in C.elegans

为验证秀丽隐杆线虫(C.elegans)sqt-1的5'端侧翼序列(5'-FSsqt-1)是否具有启动旋毛虫(T.spiralis)基因表达的功能,本研究采用PCR方法从C.elegans全基因组DNA中扩增5'-FSsqt-1,并将其克隆至以绿色荧光蛋白(GFP)为报告基因的C.elegans启动子验证载体p PD95.77中,构建重组质粒p PD-sqt-GFP。将T.spiralis表皮胶原蛋白rol6编码序列克隆至5'-FSsqt-1下游,构建重组质粒p PD-sqt-rol6-GFP。通过显微注射法将p PD-sqt-GFP和p PD-sqt-rol6-GFP分别转入C.elegans体内,进行荧光观察及western blot鉴定。结果显示,T.spiralis表皮胶原蛋白Rol6能够在C.elegans体内获得表达。研究表明,5'-FSsqt-1具有启动T.spiralis基因表达的功能,为T.spiralis基因在C.elegans体内表达奠定了基础。

The objective of this study is to verify the function of 5’ flanking sequence from Caenorhabditis elegans sqt-1 gene（5’-FSsqt-1） to promote the expression of Trichinella spiralis gene in C.elegans. 5’-FSsqt-1 were amplified by PCR from genomic DNA of C.elegans and cloned into C.elegans special vector pPD95.77 with gfp reporter gene to construct the recombinant plasmid pPD-sqt-GFP. Then the rol6 coding sequence of T.spiralis cuticle collagen was amplified and cloned under the 5’-FSsqt-1 to construct the recombinant plasmid pPD-sqt-rol6-GFP. These recombinant plasmids were transferred into C.elegans by microin- jection to assay the target protein expression by fluorescence observation and western blot. The result showed that rol6 coding sequence of T.spiralis cuticle collagen was able to be expressed in C.elegans, and the result also proved that 5’-FSsqt-1 was capable to promote the expression of T.spiralis gene in C.elegans. Those data provided a basis for expression of T.spiralis gene in C.elegans.