黑脊倒刺鲃vasa同源基因的克隆及表达分析  

cDNA cloning and expression analysis of a vasa-like gene in Spinibarbus caldwelli

作　　者：唐良华[1,2] 苏敏[1,2] 吕博彦[1,2] 詹冰津[1,2] 池丽影[1,2] 祝玲[1,2] 周琼[1,2] 

机构地区：[1]福建师范大学福建省发育与神经生物学重点实验室,福建福州350108 [2]福建师范大学生命科学学院,福建福州350108

出　　处：《水产学报》2012年第6期868-878,共11页Journal of Fisheries of China

基　　金：国家自然科学基金项目(81072616);福建省自然科学基金计划资助项目(2011J01149);福建省教育厅科技基金项目(JA09063)

摘　　要：为了从分子水平上研究鱼类生殖细胞发育的机制,首次从重要的经济养殖鱼类——黑脊倒刺鲃中克隆了斑马鱼vasa的同源基因ScVHG。该cDNA长1962bp,编码654个氨基酸,氨基酸序列中含有DEAD蛋白家族特有的9个保守结构域。经比对发现,其编码的蛋白序列与斑马鱼VASA蛋白等具有高度的同源性,与斑马鱼、罗非鱼、虹鳟、银鲫、黄鳝、金鱼、金枪鱼、草鱼的VASA蛋白相似度分别为77%,77%,79%,90%,74%,89%,79%和86%。RT-PCR结果显示:在成鱼不同的组织中,ScVHG仅在精巢和卵巢中表达。RNA原位杂交结果进一步表明:在精子发生中,ScVHG只在精原细胞中表达,而在后期的精母细胞、精子细胞中均未发现明显的表达;在卵子发生中,ScVHG在卵原细胞和卵母细胞的各个时相中均有表达,在卵原细胞中表达最为强烈,而在随后各时相的卵母细胞中,阳性信号逐渐减弱。研究认为,该基因中含有的保守序列、序列的相似性以及该基因在生殖细胞中的特异性表达等结果均表明,克隆得到的ScVHG是斑马鱼vasa的同源基因,此基因可作为一种有效的分子标记,用于黑脊倒刺鲃以及其他鱼类生殖细胞发育机制的研究。In order to interpret the development mechanism of fish germ cells at a molecular level, a Spi-nibarbus caldwelli homolog of the zebrafish vasa gene, ScVHG（S. caldwelli vasa homolog gene）, was cloned and characterized for use as a molecular marker for germ cells in this species. Analysis of the nu-cleotide sequence revealed that ScVHG comprises an open reading frame of 1 932 bps encoding 644 amino acids. The deduced amino acid sequence contained nine conserved motifs belonging to the DEAD-box protein family. The S. caldwelli VASA protein sequence showed high similarity to that of zebrafish （77%）, Oreochromis niloticus（77%）, Oncorhynchus mykiss（79%）, Carassius auratus gibelio（90%）, Monopterus albus（74%）, Carassius auratus（89%） Thunnus orientalis（79%） and Ctenopharyngodon idellus（86%）. In adult tissues, the ScVHG transcripts were specifically detected in ovary and testis. In situ hybridization analysis showed that ScVHG messenger RNA （mRNA） was detected in spermatogonia, but not in sperma-tocytes, spermatoblasts and sperms; During oogenesis, ScVHG mRNA was detected in all time phases from oogonia to oocytes. The earlier phase of oogenesis, the more ScVHG mRNA molecules were detected. The positive signals of ScVHG mRNA gradually weakened in the late phase of oocytes and finally these signals gathered round the cell edge. Consequently, consensus sequences, sequence similarity, and specific local-ization of ScVHG mRNA in the germ cells all suggest that ScVHG is the S. caldwelli homolog of the ze-brafish vasa. Further, ScVHG can be used as a molecular marker for S. caldwelli germ cells.

关 键 词：黑脊倒刺鲃 VASA 原位杂交 生殖细胞 

分 类 号：Q785[生物学—分子生物学] S917[农业科学—水产科学]