三疣梭子蟹甲基转移酶2基因克隆及在胚胎、幼体和性腺发育过程中的表达分析

Molecular cloning of the DNA methyltransferase-2 gene and its expression during embryonic, larval, and gonadal development in the swimming crab Portunus trituberculatus

本研究采用SMART RACE方法克隆了三疣梭子蟹(Portunus trituberculatus)Dnmt2(Pt Dnmt2)基因。该基因c DNA全长为1291 bp,开放阅读框为1203 bp,编码400个氨基酸。结构域分析显示,PtDnmt2基因有典型的C5-DNA甲基化酶结构域。同源分析表明,PtDnmt2氨基酸序列与其他物种有较高的同源性。系统进化分析显示,三疣梭子蟹PtDnmt2氨基酸序列与罗氏沼虾(Macrobrachium rosenbergii)Dnmt2关系最近。qRT-PCR结果显示,PtDnmt2基因在三疣梭子蟹所有组织中均有表达,在卵巢中表达量显著高于其他组织(P<0.05)。PtDnmt2基因在胚胎和幼体发育过程中的表达量随发育时期而变化,其在受精卵和多细胞时期无表达,从囊胚期开始出现,随着胚胎的发育表达量逐渐上升。在性腺发育不同时期PtDnmt2基因表达量存在显著差异,其在卵巢II期表达量最高,之后逐渐下降;在精巢中该基因的表达量随着精巢的发育逐渐上升,在IV期达到峰值。本研究结果表明,PtDnmt2基因参与了三疣梭子蟹胚胎、幼体和性腺发育调控。

DNA Methyltransferase-2(Dnmt2)is one of the most widely distributed DNA methyltransferases in eukaryotes and plays important roles in eukaryotic development.In this study,the full-length cDNA of the Dnmt2 gene is first cloned from the swimming crab(Portunus trituberculatus)using rapid amplification of cDNA ends(RACE).The length of Dnmt2 cDNA is 1291 bp,and contains a 1203 bp open reading frame that is predicted to encode 400 amino acids.Structural domain analysis revealed that the protein of PtDnmt2 contains a typical methyltransferase structural domain.Homology analysis revealed that the PtDnmt2 in P.trituberculatus exhibits high identity with those in other species.Phylogenetic analysis showed that the amino acids of PtDnmt2 are in the same branch as those of Macrobrachium rosenbergii.PtDnmt2 was found to be expressed in all tested tissues,and highly expressed in ovary and heart tissues(P<0.05).PtDnmt2 showed stage-specific expression during embryonic and larval development.No expression was detected in the zygote and multicellular stages.It was,however,detected from the blastula stage,after which expression increased gradually with embryonic development.Expression of PtDnmt2 changed significantly during gonadal development.During ovarian development,the highest expression occurred in stage II after which the level dramatically decreased.During testis development,the expression of PtDnmt2 increased gradually and reached its peak in stage IV.The results suggest that PtDnmt2 is important in the regulation of embryonic,larval,and gonadal development,and will provide useful information for further research on the roles of DNA methylation in developmental regulation in P.trituberculatus.