斑节对虾细胞周期蛋白Y基因克隆与原核表达

摘要

Cyclin Y is a newly discovered cell cycle-related protein that plays an important role during embryonic de-velopment, cell cycle progression and development, and disease. Current research on cyclin Y is very active but no data have been reported on cyclin Y in crustaceans. Gonads of the black tiger shrimp, Penaeus monodon, mature if the eye stalk is removed. Thus, the full-length cyclin Y cDNA sequence from P. monodon (denoted as Pm-cyclin Y) was ob-tained using the rapid amplification of complementary DNA ends method to better understand the potential function of cyclin Y in the regulation of shrimp reproduction. The full-length cDNA sequence was 1578 bp and contained 108 bp 5′untranslated region (UTR) and a 439 bp 3′UTR. The open reading frame was 1029 bp and coded 342 amino acids (aa). A bioinformatics analysis showed that the amino acid coding sequence had a conserved cyclin box and the ho-mologous protein box structure domain was 172–257 aa. The predicted molecular weight was about 38.7 kD, and the theoretical isoelectric point was 6.64. A real-time quantitative polymerase chain reaction analysis detected significantly higher Pm-cyclin Y mRNA expression in the ovary than that in other tissues. Pm-cyclin Y mRNA was expressed in the ovary at five different developmental stages, and the expression level was highest during phase III. The study abtained recombinant expression Pm-cyclin Y in prokaryotes and offered theoretical basis for further research on Pm-cyclin pro-tein function. These results provide a basis for further functional studies of Pm-cyclin Y.%为了研究细胞周期蛋白Y(cyclin Y)在斑节对虾(Penaeus monodon)卵巢发育中的作用,从斑节对虾转录组数据中筛选获得cyclin Y基因部分序列,采用SMART-RACE方法克隆得到斑节对虾细胞周期蛋白Y (Pm-cyclin Y)基因cDNA全序列。Pm-cyclin Y基因cDNA全长1576 bp,其中包含108 bp的5′非编码区(5′UTR)和439 bp的3′非编码区(3′UTR)以及1029 bp的开放阅读框(ORF),可编码342个氨基酸。生物信息学分析显示,其编码的氨基酸序列有1个保守的周期蛋白框(cyclin box)同源结构域(172~257 aa),预测的分子量约为37.6 kD,理论等电点6.64。实时定量 PCR 显示其 mRNA 在卵巢的表达量显著高于其他组织(P<0.05)；并且在卵巢5个不同发育期都有表达,在III期卵巢中的表达量最高。本研究通过原核表达方法对Pm-cyclin Y进行重组表达,为其蛋白质功能方面的研究奠定了基础。