RNA干扰阔口游仆虫γ-微管蛋白基因表达的研究  

RNA Interfererence of the Expressing of γ-tubulin Gene in Euplotes eurystomus by Feeding

作　　者：曾红[1] 陈文列[2] 林能锋[3] 林如辉[2] 顾福康[4] 

机构地区：[1]福建师范大学生命科学学院,福建福州350108 [2]福建中医学院中西医结合研究院,福建福州350108 [3]福建省农业科学院生物技术研究所,福建福州350003 [4]华东师范大学生命科学学院,上海200062

出　　处：《福建师范大学学报（自然科学版）》2010年第2期88-91,共4页Journal of Fujian Normal University：Natural Science Edition

基　　金：福建省科技厅青年人才资助项目(2007F3023);福建省教育厅资助项目(JA07048)

摘　　要：根据阔口游仆虫微管蛋白基因序列设计1对引物,扩增出692bp的γ-微管蛋白基因片段,将之重组到L4440载体中,重组质粒转化到RNAase缺陷型E.coli HT115菌株中,并用IPTG诱导双链RNA的表达,将经诱导后的重组菌喂食游仆虫,诱导出RNAi表型,具体表现为虫体变小、变圆,约两周后游仆虫全部死亡.电镜观察表明,经诱导后的游仆虫部分纤毛杆横切面显示结构为9+0,部分纤毛杆膨胀变形并开始瓦解,原高度有序的微管排列结构消失.进一步证实喂食法可有效地产生RNAi,γ-微管蛋白基因沉默后游仆虫无法维持正常形态并死亡,观察到纤毛杆的超微结构发生明显的变化,由此推测γ-微管蛋白的表达和细胞调控在游仆虫细胞微管骨架的装配及其细胞生命活动中起重要作用.The 692 bp gene sequence encoding Euplotes eurystomus γ-tubulin was cloned and reconstructed to vector L4440 and then transferred to E. coli HT115. After that RNAi feeding was induced to ablate expression of γ-tubulin and corresponding RNAi phenotype was induced. That is,the cell turned smaller and round,and died about two weeks later. TEM observes indicated, some of the ciliary axonemal structures turned into 9＋0 axonemes and some of the shaft expanded and began to disorganize, and the originally highly ordered arrays of microtubule disappeared. This further proved RNAi was inducible by feeding. When γ- tubulin gene was silenced the cell could not maintain normal form and then died. Ultrastructure showed the shaft changed a lot. This could infer the expression and cellar regulation of γ-tubulin was very important in the assembly of microtubule cytoskeleton and its life.

关 键 词：游仆虫 Γ-微管蛋白 喂食法 RNAI 

分 类 号：Q245[生物学—细胞生物学]