优雅蝈螽VASA蛋白cDNA序列的克隆与生物信息学分析

Cloning and bioinformatic analysis of VASA c DNA from Gampsocleis gratiosa

Vasa基因属于DEAD-box家族,其功能主要是特定mRNA的翻译调控。在许多动物中,它都是生殖系细胞发育所必须,对生殖干细胞分化具有重要作用。为探究vasa基因在半变态类昆虫生殖系细胞发育中的作用,本研究首先从基于Illumina高通量测序平台测得的优雅蝈螽Gampsocleis gratiosa成体转录组数据中筛选出一段长度为1215 bp的vasa基因片段,进而设计引物并利用RT-PCR和RACE技术获得其c DNA序列全长,最后利用生物信息学技术进行分析。结果显示:优雅蝈螽vasa基因的c DNA序列全长3359 bp,其中,5'端非编码区82bp,3'端非编码区1306 bp,开放阅读框1971 bp编码656个氨基酸,理论蛋白相对分子量(Mw)72.3 k Da,等电点(p I)5.48。通过与Gen Bank数据库中收录的其他VASA蛋白序列比对,发现优雅蝈螽VASA蛋白具有DEAD-box蛋白家族所共有的9个保守基序,Ax TGo GKT(I)、PTRELA(Ia)、TPGR(Ib)、DEAD(Ⅱ)、SAT(Ⅲ)、LVFVE(Ⅳ)、TDVu ARGID(Ⅴ)、HRIGRTGR(Ⅵ)和Gacc Poh1Q(Q),其中,Gacc Poh1Q(Q)的第3个氨基酸残基存在显著变化,建议将Gacc Poh1Q(Q)修改为Gaxc Poh1Q(Q)。此外,优雅蝈螽VASA蛋白的N端还具有10个RG和2个RGG重复序列、起始及终止密码子附近的色氨酸(W)、C末端的7个氨基酸残基中有4个为酸性氨基酸残基(E),表明其具有ATP依赖的RNA解旋酶活性。基于氨基酸序列聚类结果显示:优雅蝈螽位于六足动物分枝末梢,与双斑蟋Gryllus bimaculatus的亲缘关系最近,这与二者的分类学地位相符。本研究表明基于短读长二代测序平台获得的转录组数据可以很好地服务于功能基因研究,所获得的优雅蝈螽vasa基因c DNA全长对于进一步深入研究VASA蛋白在半变态类昆虫生殖系细胞发育研究具有重要意义。

Vasa is a DEAD- box RNA helicase that functions in translational regulation of specific mRNAs. In many animals it is essential for germ line development and may have a more general stem cell role. In order to have a further known about its function,we conducted a study selecting the 1215 bp fragment of vasa gene from Gampsocleis gratiosa transcriptome sequencing data set. By conducting RT-PCR and RACE experiment,we get the whole sequence of its cDNA with the defined specific primer and do the preliminary bioinformatics analysis. The result shows that the whole sequence of its cDNA is3359 bp,which contains 5＇- untranslated region of 82 bp,3＇- untranslated region of 1306 bp,and an opening reading frame（ ORF） of 1971 bp encoding 656 amino acids. Predicted VASA protein sequencecontains 9 conserved regions of DEAD- box family protein,Ax TGo GKT（ I） 、PTRELA（ Ia） 、TPGR（ Ib） 、DEAD（ Ⅱ） 、SAT（ Ⅲ） 、LVFVE（ Ⅳ） 、TDVu ARGID（ Ⅴ） 、HRIGRTGR（ Ⅵ） and GaccPoh1Q（ Q）,which indicates it is with ATP relied RNase active. Through VASA protein sequences alignment,we found that the third residues of GaccPoh1Q（ Q） motif existed obvious variation,and it should be renamed Gaxc Poh1Q（ Q）. In addition,there are 10 RG repeated sequences and 2 RGG repeated sequences exist in the N terminal,7 amino acids including 4 acidic amino acids residue（ E） in the C terminal,and two Try amino acids residues（ W） near the initiation and termination codon. The phylogenetic analysis reveals that G. gratiosa belongs to the Hexapoda clade, and has a closer relationship to Gryllus bimaculatus,which also belong to order Orthoptera,suborder Ensifera. Our research also indicates transcriptome data acquired using short- read Illumina sequencing platform could significantly serve for functional gene studies of nonmodel organisms in the future. Acquired vasa gene full- long cDNA of G. gratiosa plays a significant role in further studying the function of VASA protein during the germline development of Hemimetabolous insect.