三角帆蚌中WNT4基因克隆及表达分析

为进一步了解WNT4在三角帆蚌性别决定过程中的作用，通过RACE（Rapid-amplification of cDNA ends）技术克隆获得了三角帆蚌WNT4基因的全长cDNA，共1560 bp，其中5’-UTR 396 bp、3’-UTR 24 bp，开放阅读框（ORF）为1140 bp，可以编码379个氨基酸，且该序列含有Wnt家族特有结构域。对WNT4的氨基酸序列进行同源性分析后，发现该基因在物种间的进化关系和结构功能具保守性。通过实时荧光定量PCR(qRT-PCR)分析发现WNT4基因在三角帆蚌雌雄多个组织中(外套膜、闭壳肌、鳃、性腺、斧足和肝)均有表达，表明其参与了多样的生物学过程，在腮、斧足、肝中表达量较高，在性腺、闭壳肌、斧足中雌雄间有显著性差异，且雌性三角帆蚌的性腺、闭壳肌表达量显著高于雄性。原位杂交结果显示，WNT4在雌性三角帆蚌的卵母细胞上有明显的杂交信号，推测WNT4基因与性别决定相关。

Cloning and expression analysis of WNT4 gene in the Hyriopsis cumingii

In order to further understand the role of WNT4 in the sex determination process of Hyriopsis cumingii, the full-length cDNA of WNT4 gene was cloned by RACE method(Rapid-amplification of cDNA ends), which was 1560 bp, including 5''-UTR 396 bp and 3''-UTR 24 bp. The open reading frame (ORF) was 1140 bp, encoded a putative protein of 379 amino acids that contained a Wnt family specific domain. Homology analysis of the amino acid sequence of WNT4 showed that the evolutionary relationship and structural function of the gene were conserved among species. Real-time quantitative PCR (qRT-PCR) analysis of WNT4 gene in the male and female tissues (the mantle, the adductor muscle, the gill, the gonad, the foot and the liver), indicated that it participated in diverse biological processes. It was highly expressed in gill, foot, and liver. There was significant difference between male and female in gonads, adductor muscle, and foot, and the expression of female gonads and adductor muscle was significantly higher than that of males. In situ hybridization results showed that obvious signal concentrated in the female oocyte cell. The study speculated that WNT4 gene was related to sex determination.