【摘要】 vasa基因编码DEAD-box家族成员中一种ATP依赖的RNA解旋酶,在生殖系分化过程中发挥着重要的作用。本研究采用同源克隆和cDNA末端快速扩增技术(RACE),从刺参(Apostichopus japonicus)精巢中克隆得到vasa的全长cDNA序列。该cDNA序列全长2167bp,开放阅读框1593bp,编码530个氨基酸,具有DEAD-box家族蛋白的全部9个保守域。经同源比对和系统进化分析,确定其属于DEAD-box家族的VASA亚家族成员。利用半定量RT-PCR检测,vasa mRNA专一性的在刺参性腺中表达,据此,刺参vasa基因有望用于其生殖系起源和分化的研究。更多还原

【Abstract】 The vasa gene encodes an ATP-dependent RNA helicase protein member of the DEAD-box family. The vasa mRNA is initially found as a component of germ plasm in Drosophila and specifically expressed in the germ cell lineage during its germline differentiation. Subsequently,vasa gene has been found to be only limited in the germ cell lineage in many metazoans except for Hydra and planarian. Up to now,vasa gene has been extensively used as a specific molecular marker of germ-line cells and played an important role in the research of gametogenesis and reproductive regulation. Sea cucumber (Apostichopus japonicus) is one of the most commercially important Echinodermata in China. To detect whether vasa gene is specifically expressed in the germ cell lineage in sea cucumber is very important for the development of further investigation on the germline origin and differentiation in sea cucumber. In this study,full-length cDNA sequence of Aj-vasa was cloned from testes of sea cucumber,A. japonicus, by Rapid Amplification of cDNA End (RACE) methods. The full length of Aj-vasa is 2167 bp,containing a 3’ untranslated reagion (UTR) of 449 bp,a 5’ UTR of 125 bp,and an open reading frame (ORF) of 1593 bp, encoding 530 amino acids. The putative amino acid sequence shares all the nine conserved motifs of the DEAD- box family: xYxxPTPVQ,AQTGSGKT,PTRELA,TPGRI,DEAD,SAT,LVFVE,RGLD,HRIGRTGR and GG repeats. While,multiple arginine-glycine-arginine (RGG) repeats existing in the NH2-terminal portion in other VASA-related proteins in many insects and chordates,which have the function of RNA-binding,were not found in Aj-VASA,implying that its function may be realized by space reactions among different motifs. High identity of VASA in amino acid sequence (more than 58 %) was observed between A. japonicus and the other organisms including Strongylocentrotus purpuratus through Blast analysis,indicating that the cDNA cloned in this study belonged to the subfamily of VASA. Phylogenetic tree showed that A. japonicus was closely related to S. purpuratus based on amino acid sequences of VASA. Furthermore,tissue expression of Aj-vasa was detected by semi-quantitative RT-PCR,and found it specifi cally expressed in the adult gonads of both male and female. Therefore,it was deduced primarily that the Aj-vasa may be a useful maker for investigating the germline origin and differentiation in sea cucumber A. japonicus.更多还原