虾夷马粪海胆(Strongylocentrotus intermedius)Vasa基因的克隆与表达

cDNA Cloning and Expression Analysis of Vasa Gene in Strongylocentrotus intermedius

Vasa基因是DEAD-box家族成员中的一种ATP依赖的RNA解旋酶编码基因,在生殖系分化过程中发挥重要作用。采用同源克隆和cDNA末端快速扩增技术(RACE),从虾夷马粪海胆精巢中克隆得到Vasa基因3′端cDNA序列。该3′末端cDNA长1057bp,与太平洋牡蛎、紫球海胆、非洲爪蟾、小鼠及虹鳟经同源进行比对,其中与紫球海胆的同源率最高达到95%,并确定其为DEAD-box家族的Vasa亚家族成员。利用实时定量RT-PCR检测Vasa mRNA在虾夷马粪海胆组织和胚胎发育期的表达情况,研究结果表明Vasa基因在生殖腺表达量最高,雌、雄个体生殖腺中转录水平上的表达差异明显,雄性高于雌性,差异显著(P<0.05),在肠、体腔液、肌肉、围口膜、管足中表达量低,其中体腔液表达量最低。胚胎发育期检测发现Vasa基因在16细胞期时表达量最高,16细胞期后表达量呈下降趋势,囊胚期表达量最低。试验结果为虾夷马粪海胆生殖系统起源、分化研究提供科学依据。对于虾夷马粪海胆生殖系分化途径来讲,Vasa可作为一种有利的研究工具,追溯其生殖系的起源和分化。

Vasa is a DEAD-box gene family members in an ATP dependent RNA helicase,plays an important role during reproductive differentiation.In this study the homology cloning and rapid amplification of cDNA ends（RACE）,was cloned from Strongylocentrotus intermedius male gonad,get Vasa gene 3＇end cDNA sequences.The 3＇end of the cDNA length 1 057 bp,and the Pacific oyster,Strongylocentrotus purpuratus,Xenopus,mouse and rainbow trout were compared by homology,in which the homology with the Strongylocentrotus purpuratus,the highest rate of 95%,and identified it as DEAD-box subfamily members of the Vasa family.Using real-time quantitative RT-PCR detection of Vasa mRNA in Strongylocentrotus intermedius organization and expression of embryonic development,the results show that the Vasa gene expression level in the gonads,male and female gonads in the expression of the transcription level of significant differences,males than females the difference was significant（P0.05）,in the intestine,body cavity fluid,muscle,around mouth membrane,tube full of expression,among which the lowest amount of body fluid expression.Embryonic development detected Vasa gene expression in the 16-cell stage when the highest,after the expression of 16 cells decreased the expression of the lowest blastocyst stage.The results for the origin of Strongylocentrotus intermedius reproductive system,to provide a scientific basis for differentiation.For Strongylocentrotus intermedius way in terms of reproductive differentiation,Vasa can be used as an enabling research tool,to trace its origin and differentiation of the reproductive system.