Full-long cDNA sequence cloning and bioinformatic analysis of Piwi subfamily member Giwi in the Gampsocleis gratiosa gonads

• 1、College of Life Sciences, Hebei University, Baoding 071002

Abstract：Piwi-like genes represent essential stem cell genes in diverse multicellular organisms. However, it has not been reported in hemimetabolous insect. 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 3,462bp, which consisted of a 2742 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 kDa 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 a 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, which 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.

Keywords： Zoology Gampsocleis gratiosa Germline Stem Cell Piwi Transcriptome cDNA 3D structure