牙鲆dazl基因的克隆与表达分析  

Molecular cloning and expression analysis of dazl gene in Paralichthys olivaceus

作　　者：孙近近 张俊玲[1] 孙文慧[1] 施志仪[1] 

机构地区：[1]上海海洋大学水产与生命学院农业部淡水水产种质资源重点实验室,上海201306

出　　处：《海洋渔业》2016年第4期391-399,共9页Marine Fisheries

基　　金：国家自然科学基金青年项目(41306128);国家自然科学基金面上项目(30271017)

摘　　要：dazl(deleted in azoospermia-like)基因是多种物种精子发生的重要调控因子,为探讨该基因在牙鲆(Paralichthys olivaceus)性腺发育分化中的作用,本研究采用同源克隆和RACE技术克隆了牙鲆dazl基因的cDNA序列,利用生物信息学方法分析了其序列结构,并利用实时荧光定量PCR技术检测了该基因在牙鲆成体组织和早期发育阶段的表达情况。结果发现:牙鲆dazl cDNA序列全长2 031 bp,包括105 bp的5'端非翻译区,1 275 bp的3'端非翻译区和编码217个氨基酸残基的651 bp开放阅读框;氨基酸同源序列比对显示该序列存在一个高度保守的RRM(RNA recognition motif)结构域,且该结构域在鱼类中具有100%的序列一致性;荧光定量PCR结果显示dazl基因主要在牙鲆性腺中表达,且在精巢中的表达高于卵巢;在牙鲆早期发育阶段,dazl基因在未受精卵、受精卵及囊胚期均有较高的转录本,而在原肠胚期表达开始降低,至孵化后3 d一直保持较低的水平,这些丰富表达的母源性dazl mRNA可能参与了胚胎发育中原始生殖细胞的形成。本研究为进一步阐明dazl基因在牙鲆生殖发育中的功能奠定了基础。dazl （deleted in azoospermia-like ） gene is an important regulator of a variety of species of spermatogenesis. To explore the role of dazl gene in gonad differentiation and development of Paralichthys olivaceus, the full-length cDNA of dazl gene was cloned using homology cloning and RACE technology, its sequence structure was analyzed by bioinformatics method, and its gene expression in different adult tissues and during early development of P. olivaceus was studied using real-time PCR technology. The results showed that the full-length cDNA sequence of P. olivaceus dazl was 2 031 bp, including 105 bp of 5＇untranslated region, 1 275 bp of 3＇ untranslated region and 651 bp of open reading frame encoding 217 amino acid residues. The dazl had a highly conserved RNA recognition motif, which shared 100% sequence similarities among fish species by amino acid sequence homology comparison. RT-qPCR results showed that the dazl gene was mainly expressed in gonad, and it had a higher expression level in testis than in ovary. During the early development, the dazl gene had higher transcriptions in the unfertilized egg, fertilized egg and blastocysts, and its expression began to reduce at gastrula stage, and remained at a low level until 3 days after hatching. These results suggest that the abundant maternal dazl mRNA may be involved in the formation of primitive germ cells in the embryonic development. This study provides a basis to further clarify the role of dazl gene in P. olivaceus reproductive development.

关 键 词：牙鲆 DAZL CDNA克隆 基因表达 

分 类 号：Q782[生物学—分子生物学]