title: Cloning, expression and methylation analysis of piwil2 in half-smooth tongue sole (Cynoglossus semilaevis).
abstract: piwi is an important regulator gene in germ cell division during spermatogenesis. piwi homologous genes are involved in gametogenesis and germline specification, and knocking down these genes could affect germ cell meiotic progression. To understand the function of piwi-related genes in spermatogenesis, we cloned a Piwi-subfamily member (piwil2 gene) from the gonad of Cynoglossus semilaevis. The full-length of piwil2 cDNA was 3314bp, including a 3162bp open reading frame (ORF), a 60bp 5'-UTR, along with a 92bp 3'-UTR, and encoded a predicted protein of 1053 amino acid residues. Phylogenetic analysis showed that the PIWIL2 putative protein belonged to the Argonaute protein family, and Piwi-subfamily, with typical PAZ and Piwi domains. Ultrathin sections of different gonadal stages, and real-time quantitative PCR showed that the relative expression of the piwil2 gene couldn't be detected until day 95 (95days) larvae, when germ cell divided rapidly in C. semilaevis. The piwil2 transcripts were more abundant in gonads of males and neo-males than in females, and weakly expressed in other tissues and organs. Compared with the relative expression of piwil2 in gonads, the CpG methylation levels were significantly higher in females. Chromosomal fluorescence in situ hybridization (FISH) showed that the piwil2 gene was located on the Z sex chromosome of C. semilaevis. These results suggest that piwil2 plays an important role in spermatogenesis of C. semilaevis.
authors.affiliation: College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China; Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China.
authors.forename: Liyan
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; Fisheries College, Ocean University of China, 5 Yushan Road, Qingdao 266003, China.
authors.forename: Wanjun
authors.initials: W
authors.lastname: Liu
authors.affiliation: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China.
authors.forename: Changwei
authors.initials: C
authors.lastname: Shao
authors.affiliation: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Zhang
authors.affiliation: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China.
authors.forename: Hailong
authors.initials: H
authors.lastname: Li
authors.affiliation: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Liu
authors.affiliation: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; Fisheries College, Ocean University of China, 5 Yushan Road, Qingdao 266003, China.
authors.forename: Zhongdian
authors.initials: Z
authors.lastname: Dong
authors.affiliation: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; Fisheries College, Ocean University of China, 5 Yushan Road, Qingdao 266003, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Qi
authors.affiliation: College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Zhao
authors.affiliation: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China. Electronic address: chensl@ysfri.ac.cn.
authors.forename: Songlin
authors.initials: S
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D060565:Argonaute Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D019175:DNA Methylation; D005260:Female; D005412:Flatfishes / Q000145:classification / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D005786:Gene Expression Regulation; D006066:Gonads / Q000166:cytology; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D010802:Phylogeny; D012730:Sex Chromosomes / Q000235:genetics; D013091:Spermatogenesis / Q000235:genetics
chemical_list: D060565:Argonaute Proteins
keywords: Histological observations; Phylogenetic analysis; RACE; RT-qPCR; Spermatogenesis