半滑舌鳎 WT1a 基因的克隆与性别分化期的表达分析

摘要

从半滑舌鳎(Cynoglossus semilaevis)全基因组序列中获得肿瘤抑制基因 WT1a 的部分序列,然后利用RACE 技术克隆了 WT1a 基因的全长,运用组织学方法,对半滑舌鳎性腺分化早期的组织切片进行了观察,并分析了该阶段WT1a 的表达特征。克隆结果显示, WT1a 基因cDNA全长为1886 bp,开放阅读框(ORF)长为1470 bp,编码490个氨基酸,并且包含 KTS 三肽,5′-UTR 和3′-UTR 分别长为245 bp 和171 bp。在 ORF 末端发现2个微卫星(SSR)序列和2个跨膜螺旋区域,而在其他物种中未发现这些区域。氨基酸序列分析显示, WT1a 基因的保守性很高,与人的同源性高达90%。通过组织切片观察发现,该批半滑舌鳎在56 d 时性腺开始分化,并且卵巢分化早于精巢。荧光实时定量分析结果显示, WT1a 在半滑舌鳎成鱼性腺中表达量显著高于其他各组织,在肝、肾、脾、心脏等组织中有微量表达,并且在成鱼性腺中,雄鱼表达量显著高于雌鱼,雌鱼显著高于伪雄鱼。在胚胎发育各时期以及初出膜后WT1a的表达结果显示,在12 h (原肠早期)显著升高,20 h (神经胚期)达到最高。在性腺分化早期雌雄性腺中的表达结果显示, WT1a 在雌雄半滑舌鳎16~66 d 的性腺中表达量持续平稳升高,在性腺分化时期表达量并没有特殊的变化。这些研究结果说明, WT1a 基因是半滑舌鳎性别相关基因,并在半滑舌鳎的性腺分化、发育过程中持续表达,但可能对性腺的分化过程并不起决定作用。%Half-smooth tongue sole (Cynoglossus semilaevis) females grow two to three times faster than males. Thus, the development of an all-female stock would be of significant benefit for aquaculture. To develop an all-female C. semilaevis stock, investigation of sex determination mechanisms is necessary. The Wilms’ tumor suppressor gene (WT1) encodes a zinc-finger transcription factor that plays an essential role in organ development, most notably in the kidney and gonad. Despite its importance for sex determination, information on WT1 expres-sion regulation is scarce. Here, the full-length cDNA of a C. semilaevis WT1a gene was obtained by RACE-cloning. Sequence analysis revealed that the full-length cDNA was 1 886 bp containing a 1 470 bp open reading frame (ORF), a 245 bp 5′-UTR, and a 171 bp 3′-UTR. This sequence encoded 489 amino acids containing a KTS alternative splice. Two SSR sequences and two transmembrane helix regions were found in the end of the OFR. We infer that they are C. semilaevis specific sequences. Alignment analysis revealed that the C. semilaevis WT1a gene was highly homologous to that of Danio rerio and Oncorhynchus mykiss. An NJ-phylogenetic tree indicated that the fish WT1a gene is more closely related to the WT1 gene of other species than it is to the fish WT1b gene. Real-time quantitative PCR results revealed that WT1a was expressed in the six tissues types exam-ined but was far higher in the gonad. Notably, expression in the testis was significantly higher than that in the ovaries and gonads of sex-reversal female fish. It was lowest in the gonad of sex-reversal female fish. Therefore, we can conclude that WT1a is a sex-related gene. The gene was expressed in all of the developmental stages ana-lyzed, from early embryo to 66-day-old fry. Its expression level was highest at neurula stage (20 h) and gently in 16-66 days, however, no abnormalities were observed in the key gonad differentiation stages. Therefore, WT1a may be not the key gene controlling gonad differentiation. Our results provide a foundation for the development of an all-female C. semilaevis stock and future investigations on the sex determination mechanism. Furthermore, we have provided a good method for identifying the key gene controling gonad differentiation.