圆斑星鲽(Verasper variegatus)wt1基因的克隆与表达分析

CLONING AND EXPRESSION OF THE WT1 GENE IN SPOTTED HALIBUT(VERASPER VARIEGATUS)

圆斑星鲽(Verasper variegatus)雌鱼生长快,成熟雌鱼个体大小是雄鱼的2倍以上,开展性别相关基因的功能研究,对于探究圆斑星鲽性别决定机制,建立单性培育技术具有重要意义。本研究获得了wt1a及wt1b两个同源基因,wt1a基因全长为3263bp,预测开放阅读框(ORF)长为1245bp,编码415个氨基酸,5′-UTR和3′-UTR分别长372bp和1640bp;wt1b基因全长为2312bp,预测开放阅读框(ORF)长为1281bp,编码427个氨基酸,5’-UTR和3’-UTR分别长369bp和659bp。wt1a基因编码氨基酸分子量为46.2kDa,理论等电点为9.24,无跨膜结构及信号肽,在ORF末端有4个锌指结构,编码KTS三肽;wt1b基因编码氨基酸分子量为46.95kDa,理论等电点为8.99,无跨膜结构及信号肽,在ORF末端有4个锌指结构,并且编码KTS三肽。基因表达结果表明:wt1a和wt1b基因主要在圆斑星鲽性腺中表达,精巢的表达高于卵巢,肾脏的表达量显著高于其他组织,推测wt1a基因和wt1b基因在性腺和肾脏发育过程及功能方面均发挥重要作用;在早期发育阶段,wt1a基因在原肠期之前微弱表达,从原肠早期开始逐渐上升至神经胚期表达量达到最高,之后逐渐下降,直至孵化阶段,推测wt1a基因在圆斑星鲽原始生殖细胞分化过程及性腺发育中发挥重要作用。

The spotted halibut(Verasper variegatus)is a rare and valuable marine fish species that inhabits the coast of the northern China.Mature females are much larger than males because of their faster growth.It will create substantial economic benefits to establish an all-female breeding technique for V.variegatus.A better understanding of sex-related genes will contribute to the improvement of a single-sex breeding technique.In this study,we successfully isolated the wt1 gene of V.variegatus,which is named Vvwt1a and Vvwt1b.The total length of wt1a was 3263bp,including a 1245bp open reading frame(ORF),encoding 415 amino acids,the 5′UTR was 372bp and the 3′UTR was 1640bp.The total length of wt1b was 2312bp,including a 1281bp open reading frame(ORF),encoding 427 amino acids,the 5′UTR was 369bp and the 3′UTR was 659bp.Through biological analysis,the wt1a gene encoded amino acids,with a predicted molecular weight of 46.2kDa and an isoelectric point of 9.24,no transmembrane structure or signal peptide site was detected,there were four zinc finger structures at the end of ORF and encode KTS.The wt1b gene encoded amino acids,with a predicted molecular weight of 46.95kDa and an isoelectric point of 8.99,no transmembrane structure or signal peptide site was detected,there were four zinc finger structures at the end of ORF and encode KTS.Real-time fluorescence quantitative PCR technique was used to analyze the expression patterns of the wt1 gene at different stages of embryo and larvae.The results show that the expression level of wt1a and wt1b genes in gonads was significantly higher than other tissues’,and the expression level in the testis was significantly higher than in the ovary.Therefore,we speculated that wt1a and wt1b genes play important roles in gonad and kidney development and function.In the early stage of development,wt1a gene weakly expressed before the early gastrula,then the expression level gradually rose to the highest level in the neural embryo stage,followed by a decline until the hatching stage.It is speculated that wt1a gene might played a role in the differentiation process of primitive germ cells and gonad development.