关键词: 优雅蝈螽, 生殖干细胞, Piwi, 转录组, cDNA, 3D, 结构

Abstract: In order to further research the role of piwi in the stem cell self-renewal and germline development of hemimetabolous insect. To obtain the full-length of piwi homolog giwi, we first picked out piwi homolog fragments in the Gampsocleis 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 full-long of giwi 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.

Key words: Gampsocleis gratiosa, Germline stem cell, Piwi, Transcriptome cDNA, 3D structure