虾夷马粪海胆硬脂酰辅酶A去饱和酶和蛋白酪氨酸磷酸酶基因的克隆与表达

摘要

采用同源克隆方法,结合cDNA末端快速扩增(RACE)技术,成功克隆了虾夷马粪海胆Strongylocentrotus intermedius硬脂酰辅酶A去饱和酶(Stearoyl-coenzyme A desaturase,SCD)基因和蛋白酪氨酸磷酸酶(Protein tyrosine phosphatase 1B,PTP1B)基因,并对其结构进行了分析,同时采用实时定量PCR方法对这两个基因在海胆胚胎不同发育时期的表达进行了研究.结果表明:SCD基因的cDNA序列全长为1 934bp(Genbank登录号为HM208174),开放阅读框819 bp,编码273个氨基酸残基,存在跨膜结构和信号肽,为分泌性蛋白;实时定量PCR检测显示,SCD基因在虾夷马粪海胆八腕期表达量最高,在2细胞时期表达量最低.PTP1B基因的cDNA序列全长为1 249 bp(Genbank登录号为HM208173),开放阅读框657 bp,编码219个氨基酸残基,存在跨膜结构和信号肽,为分泌性蛋白;PTP1B基因在海胆2细胞时期表达量最高,在八腕期表达量最低.本研究为在分子水平上探讨海胆脂代谢过程中的基因功能提供了科学依据.%Stearoyl - coenzyme A desaturase ( SCD) and Protein tyrosine phosphatase 1B ( PTP1B ) genes were cloned in sea urchin Strongylocentrotus intermedium by homology - base technique and RACE technique, and the structures of these genes were studied. A quantitative reverse transcriptase real-time PCR assay was developed to assess mRNA expression of these genes in different periods of embryonic development. SCD ( Genbank No. HM208174) was found to be secreted protein whose full-length cDNA was 1 934 bp with an ORF of 819 bp enco-ding 273 amino acids with transmembrane domain, signal peptide. Higher-level mRNA expression of SCD was de-tected in the periods of 8-arm stage. PTP1B (Genbank No. HM208173) as secreted protein had full-length cDNA of 1 249 bp with an ORF of 657 bp encoding 219 amino acids with transmembrane domain, signal peptide. The RT -PCR detection revealed that the maximal expression level of PTP1B was observed in 2-cell stage, while the mini-mal level expression was found in 8-arm stage. The findings provided the foundation for understanding of the func-tion of genes in the sea urchin metabolic processes at the molecular level.