日本血吸虫Bantam miRNA靶基因Rho相关GTP结合蛋白对雌虫卵巢发育影响的分析

Functional Analysis of A Rho Related GTP Binding Protein of BANTAM miRNA Target in Schistosoma japonicum

为鉴定Rho相关GTP结合蛋白为日本血吸虫Bantam miRNA的靶基因,并初步研究该基因的功能。利用RNAhybrid软件分析Bantam miRNA的靶基因,用3’RACE技术扩增获得靶基因与Bantammi RNA的互作区域,利用荧光素酶报告基因验证靶基因与miRNA的互作,qPCR分析日本血吸虫Rho相关GTP结合蛋白基因在雌虫不同部位的转录情况,再利用RNAi技术初步研究该基因的功能。结果发现:共转染含有与Bantam miRNA互作区域的Rho相关GTP结合蛋白的重组质粒和Bantam miRNA mimics可显著抑制荧光素酶的表达;qPCR分析表明日本血吸虫Rho相关GTP结合蛋白在雌虫的上部、卵巢、卵黄腺部均有转录,且在卵巢转录最高。RNAi抑制Rho相关GTP结合蛋白的表达时发现,虫体卵巢结构异常,出现裂纹状空泡,并且产卵量下降。Rho相关GTP结合蛋白(TNN14903.1)为血吸虫Bantam mi RNA的靶基因,干扰该基因的表达可导致日本血吸虫卵巢结构异常。

To identify the Rho related GTP binding protein as the target gene of Bantam miRNA of Schistosoma japonicum and to study its function.Analysis of target genes of Bantam miRNA by RNA hybrid software,the predictively binding region of target gene with Bantam miRNA was amplified by 3'RACE.Using luciferase reporter assay the interaction between target gene and Bantam miRNA was verified.The transcript level of the Rho related GTP binding protein of Schistosoma japonicum in different stages and different parts of female worms were analyzed by qPCR,and the function of the target gene was evaluated by RNAi.Co-transfection of recombinant plasmid containing the region of Rho related GTP binding protein interacting with Bantam miRNA mimics and Bantam miRNA mimics significantly inhibited the expression of luciferase.qPCR analysis showed that the Rho related GTP binding protein of Schistosoma japonicum was transcribed in the upper part of female,ovary and yolk gland,and the highest transcription was found in ovary.RNAi inhibited the expression of Rho related GTP binding protein at the transcript level,resulting in abnormal ovarian structure,crack like vacuoles and decreased fecundity.Rho related GTP binding protein(TNN14903.1) is the target gene of Bantam miRNA in Schistosoma japonicum,and blocking the expression of this protein can lead to the structural abnormality of ovary in Schistosoma japonicum.