三角帆蚌cyclin B基因克隆及功能

通过现代分子生物学技术,从三角帆蚌(Hyriopsis cumingii)中克隆了细胞周期蛋白B(cyclin B)基因的cDNA,得到长度为1 024 bp的序列信息,包括768 bp的ORF,197 bp的3′-UTR,编码255个Aa。进一步分析表明,三角帆蚌cyclin B基因与牡蛎、扇贝具有较高的相似性,其氨基酸序列中具有1个典型的周期蛋白框(cyclin-box),并存在2个周期蛋白依赖性蛋白激酶(CDK)的作用位点。通过RT-qPCR研究显示:三角帆蚌cyclin B基因在性腺中的表达最高,在雌性中的表达量显著高于雄性(P0.05);而cyclin B基因在其他组织中的表达量均显著低于性腺(P0.05),且无显著雌雄差异(P0.05);沉默cyclin B基因后,除外套膜外,其在性腺和鳃的表达显著降低(P0.05),表明RNA干扰(RNAi)对三角帆蚌不同组织具有不同的沉默效果。流式细胞仪测定RNAi后细胞的分裂时相变化,发现性腺和鳃细胞中G2/M期占比下降,表明cyclin B基因在三角帆蚌中能调控细胞分裂。初步研究了三角帆蚌cyclin B基因及其功能,为三角帆蚌细胞体外建系奠定分子基础。

Expression and function of cyclin B gene in Hyriopsis cumingii

The cell division of pearl membrane is slow in vitro. The current regulation mechanism is still unclear, which restricts the development of new pearl cultivation technology. In this study, the cyclin B cDNA was cloned from Hyriopsis cumingii by modern molecular biology techniques, and the sequence length is 1024 bp, including 768 bp of ORF, 197 bp of 3"-UTR, encoding 255 Aa. Further analysis showed that cyclin B has a high similarity with oysters and scallops. It has a typical cyclin box in its amino acid sequence and there were two downstream cyclin-dependent protein kinases (CDK). RT-Q PCR showed that the expression of cyclin B was the highest in the gonads, and the expression level of cyclin B in females was significantly higher than that in males (P<0.05). The expression of cyclin B in other tissues was significantly lower than that in gonad (P<0.05), and there was no significant difference between male and female (P>0.05).After RNA interference (RNAi), the expression of gonad and gill was significantly decreased except for the mantle (P<0.05), indicating RNAi is not systematic in Hyriopsis cumingii. The phase changes of cells after RNAi were determined by flow cytometry. It was found that the proportion of G2/M phase in gonad and gill cells decreased, indicating that cell division can be regulated by cyclin B in Hyriopsis cumingii. In this study, cyclin B gene and its preliminary functions were studied, and it laid a molecular basis for further regulation of mantle cell growth in vitro.