细菌介导的发夹结构双链RNA对秀丽小杆线虫中par-1基因的RNA干扰  

Double-stranded RNA expressed by hairpin structures induced RNA interference of the par-1 gene in the nematode Caenorhabditis elegans

作　　者：高雅君[1] 杨玉荣[1] 

机构地区：[1]细胞生物学与肿瘤细胞工程教育部重点实验室厦门大学生命科学学院,福建厦门361005

出　　处：《动物学报》2007年第3期545-551,共7页ACTA ZOOLOGICA SINICA

基　　金：国家自然科学基金资助项目(No.30370695)~~

摘　　要：秀丽小杆线虫(Caenorhabditis elegans)是一种重要的模式生物,目前在C.elegans中发现的许多基因在序列和功能上与哺乳动物的基因有很高的同源性,对其基因功能的研究有助于阐明哺乳动物的基因功能。为检测细菌介导的通过发夹结构表达双链RNA(dsRNA)的RNA干扰(RNAi)在C.elegans中的作用效果,我们设计并构建了可以转录表达母源极性基因par-1的发夹dsRNA的RNAi载体,并转入E.coli HT115中,经过IPTG诱导后浓缩。浓缩菌液在25℃下喂食C.elegans48h后,观察C.elegans胚胎发育情况,同时以含有空载体的菌液作为对照,并与par-1突变体及野生型比较。结果显示:发夹结构表达的dsRNA对par-1基因进行RNAi后,虫体内par-1 mRNA几乎消失,C.elegans早期胚胎分裂不对称性丧失,par-1(RNAi)干扰率在95%以上。The nematode Caenorhabditis elegans is an important model for genetic studies and many of its genes are homologous with those of other （invertebrate and vertebrate） animals. This paper reports a method of bacteria-mediated RNA interference induced by hairpin dsRNA expression vector, targeting the par- 1 gene in C. elegans. The hairpin dsRNA expression vector was constructed, based upon pBluescript Sk （ ＋ ） and transferred into E. coil HT115. Development of C. elegans embryos was observed after feeding with HT115 bacteria which can express par-1 hairpin dsRNA. After feeding with HT115, for 48 h at 25℃, bacteria-mediated expression of par-1 RNAi occurred at 95% efficiency. Bacteria-mediated par-1 RNAi resulted in the loss of asymmetric cleavage of the early C. elegans embryo. RT-PCR results showed that levels of par- 1 mRNA were almost undetectable in RNAi worms. This approach, using bacteria-mediated RNAi, may now be applied to further studies of gene expression in transgenic C. elegans [ Acta Zoologica Sinica 53 （3） ： 545 - 551, 2007].

关 键 词：秀丽小杆线虫 PAR-1 发夹结构 RNAI 克隆 构建 

分 类 号：Q75[生物学—分子生物学]