优雅蝈螽卵巢Piwi蛋白亚家族成员GiwicDNA序列全长的克隆与生物信息学分析  

Full-long cDNA Sequence Cloning and Bioinformatic Analysis of Piwi Subfamily Member Giwi in the Gampsocleis gratiosa Gonads

作　　者：刘静[1] 周志军[1] 常岩林[1] 

机构地区：[1]河北大学生命科学学院,保定071002

出　　处：《生物技术通报》2013年第2期111-117,共7页Biotechnology Bulletin

基　　金：河北省自然科学基金项目(C2012201049);教育部高等学校博士学科点专项科研基金项目(20101301120006)

摘　　要：旨在进一步研究piwi基因在半变态昆虫干细胞自我更新和生殖系细胞发育中的作用。首先对实验室前期测定的优雅蝈螽(Gampsocleis gratiosa)转录组数据进行搜索,成功挑选出piwi基因的同源体,命名为giwi。通过设计特异性引物,采用巢式RT-PCR和RACE末端扩增技术克隆获得该基因cDNA序列。序列分析表明,优雅蝈螽giwi基因cDNA序列全长为3 462 bp,包含1个完整的开放阅读框2 742 bp,编码913个氨基酸,以及一个长度为111 bp的5'端非编码区和609 bp的3'端非编码区。预测的理论蛋白分子量为102.7 kD,等电点为9.55。Giwi蛋白具备Piwi蛋白亚家族全部特征,C端的保守PIWI结构域,中部的保守PAZ结构域和N端可变结构域。同源比对分析得到PIWI结构域具有类似RNA酶H活性中心的DDH三联催化模体,暗示该蛋白具有切割活性。系统发育分析指出昆虫的Piwi和Aubergine可能源于远古基因的重复事件。通过二代测序技术获得的转录组数据可以很好地服务于未来的功能基因研究。In order to further research the role ofpiwi in the stem cell self-renewal and germline development of hemimetabolous insect. To obtain the full-length ofpiwi homolog giwi, we first picked out piwi homolog fragments in the C, ampsocleis gratiosa transcriptome sequences, which had been sequenced by our laboratory and not release to the public database, and then designed specific primers for nested RT-PCR partial amplification and rapid amplification of cDNA ends （ RACE ） complete 5＇ and 3＇sequence. Sequences analysis showed that the fulMong ofgiwi gene cDNA is 3 462 bp, which consisted of a 2 742 bp open reading frame （ ORF ） encoding 913 amino acids, a 111 bp 5＇-untranslated region （ 5＇UTR ） and a 609 bp 3＇-untranslated region （ 3＇UTR ） . The molecular weight of Giwi has been predicated to be about 102.7 kD and pI 9.55 in theory. Giwi contains the signature motifs of the piwi protein subfamily, including a C-terminal PIWI domain, a centrally located PAZ domain, and an N-terminal variable domain. The homology blast analysis showed that there is a catalytic triad ＂Asp-Asp-His＂ motif in the PIWI domain which is similar to the RNase H active center, implying possessing slicer activity of this protein. Phylogenetic analysis showed that appearance of either Piwi or Aubergine in insect might due to an ancient gene duplication event. Transcriptome resources generated based on next-generation sequencing can greatly benefit future gene cloning.

关 键 词：优雅蝈螽 生殖干细胞 PIWI 转录组 CDNA 3D结构 

分 类 号：Q78[生物学—分子生物学]