家蚕微孢子虫的一个Dicer相似蛋白基因的鉴定及系统进化和转录活性分析

Identification,Evolutionary and Transcriptional Activity Analyses of a Dicer-like Gene in Microsporidium Nosema bombycis

Dicer是一种具有RNaseⅢ酶活性的蛋白质,参与RNAi起始阶段siRNA和miRNA的形成,为RNA诱导沉默复合物（RISC）形成的关键因子之一,在基因的转录后调控中扮演重要角色。采用生物信息学方法从家蚕微孢子虫（Nosema bombycis）全基因组数据中鉴定到一个Dicer相似蛋白（Dicer-like）基因,命名为Nb DCL（Gen Bank登录号：EOB15391）。预测Nb DCL的二级结构中含有RNaseⅢ结构域和dsRNA结合结构域。基于Dicer-like基因的系统进化分析显示,家蚕微孢子虫与柞蚕微孢子虫（Nosema pernyi）的亲缘关系最近,并与真菌形成姊妹枝,说明微孢子虫Dicer-like与真菌的同源基因来自共同的祖先。基因组共线性分析显示,Dicer-like基因及其两侧基因在微孢子虫基因组中的排列顺序具有显著的保守性,但兔脑炎微孢子虫（Encephalitozoon cuniculi）和比氏肠道微孢子虫（Enterocytozoon bieneusi）基因组中的Dicer-like基因在物种分化过程中发生了丢失。对Dicer-like基因和Agronaute相似蛋白基因、转座子的共存分析表明,家蚕微孢子虫等Nosema属微孢子虫均保留了古老的RNAi作用机制。RT-PCR检测被家蚕微孢子虫感染48-240 h的家蚕幼虫中肠中Nb DCL基因均有转录。研究结果为进一步探究家蚕微孢子虫RNAi的调控机制提供了有益线索。

Dicer,a protein with RNase Ⅲ activity involved in the formation of siRNA or miRNA in the initial stage of RNA interference（ RNAi）,is one of the key factors in the formation of RNA induced silencing complex（ RISC）. It plays an important role in post-transcriptional regulation of gene expression. One Dicer-like gene,named as Nb DCL（ Gen Bank accession No. EOB15391）,was firstly identified in the genomic sequences of Nosema bombycis by means of bioinformatics＇ analysis. Protein secondary structure prediction showed that Nb DCL possessed RNase Ⅲ domain and dsRNA binding domain. Phylogenetic analysis based on Dicer-like gene showed that N. bombycis had close relationship with Nosema pernyi,and formed a monophyletic clade with fungus,which suggested that Dicer-like genes in microsporidia and homologous gene in fungi derived from a common ancestor. Genomic synteny analysis revealed that locations of Dicer-like and its flanking genes were highly conserved in microsporidian genomes. However,Dicerlike gene had been lost during the evolution of Encephali-tozoon cuniculi and Enterocytozoon bieneusi. Moreover,RNAi mechanism was retained in all microsporodia of Nosema genus as shown in co-existence analysis of Dicer-like genes with Agronaute-like gene and Tranposon. Nb DCL transcription was detected consecutively by RT-PCR from 48 to 240 h post infection with N. bombycis in silkworm larval midgut. These research results provide useful clues for further exploration on regulatory mechanism of N. bombycis RNAi.