斑节对虾细胞周期蛋白E基因的克隆与表达分析

Molecular cloning and expression analysis of the cyclin E gene from black tiger shrimp Penaeus monodon

为了解细胞周期蛋白E(cyclin E)基因在斑节对虾(Penaeus monodon)发育过程中的作用,构建和测序了斑节对虾全组织cDNA文库,获得了斑节对虾细胞周期蛋白(Pmcyclin)E的部分表达序列。通过RACE技术克隆得到1条全长1 706 bp的cDNA序列。Pmcyclin E开放阅读框(ORF)为1 263 bp,编码420个氨基酸。生物信息学分析表明,该氨基酸序列含有周期蛋白家族特有的CYCLIN保守区并含有N-糖基化位点及磷酸化位点。经BLAST同源性分析表明,该基因编码蛋白与红火蚁(Solenopsis invicta)、切叶蜂(Megachile rotundata)等多种节肢动物细胞周期蛋白E有很高的同源性。组织分布分析表明,该基因在斑节对虾精巢、卵巢、肠、脑、肝胰腺、胃和心脏组织中均有表达,其中卵巢中表达量较高,而心脏、胃及肝胰腺中表达量相对较低。分别对蜕皮抑制激素(MIH)注射和眼柄切除的对虾进行表达分析,发现眼柄切除后,Pmcyclin E大量表达,而注射MIH后细胞Pmcyclin E的表达量相对降低。结果表明,细胞cyclin E基因在斑节对虾卵巢发育过程中起重要作用,且眼柄切除对细胞cyclin E的表达具有影响,该结果为进一步探究斑节对虾卵巢的发育机理提供了理论依据。

Cyclins are the positive regulatory subunits of cyclin-dependent kinases （CDK）. They play a critical role in the coordination of the eucaryotic cell cycle by binding to the catalytic subunits of CDKs. They share distant sequence homology over a 100 amino acid region called the cyclin box and have been classified into two phy-logenetically divergent subfamilies. The first contains A, B, D, E, F, and G cyclins and the second contains the C and H cyclins. Shrimp culture is limited by a lack of methods to breed adults in captivity. This is because female shrimp are unable to reach ovarian maturity without intervention using destructive, expensive, or time consuming methods such as eyestalk-ablation. Thus, there is a need to better understand the mechanism of ovarian develop-ment. Cyclin E is a cell cycle regulatory factor that plays a very important role in cell proliferation. To better un-derstand the potential function of cell cyclin E in regulating shrimp reproduction, the full-length cDNA sequence of cell cyclin E from black tiger shrimps Penaeus monodon （denoted as Pmcyclin E） was obtained by high throughput transcriptome sequencing and RACE. The cDNA of Pmcyclin E was 1 706 bp, including a 132 bp 5′-terminal un-translated region （cUTR）, a 311 bp 3′UTR with a poly （A） tail, and an open reading frame （ORF） of 1 263 bp encoding a polypeptide of 420 amino acids with a predicted molecular weight of 47.9 kD and pre-dicted pI of 5.85. The results of Blast and phylogenetic analyses suggest that Pmcyclin E is a new member of the shrimp cell cyclin E family. We measured mRNA expression of Pmcyclin E in nine tissues by real-time PCR. Ex-pression was highest in the ovary and eyestalk, moderate in the intestine, brain, and muscle, and very low in the stomach and hepatopancreas. Pmcyclin E expression in the ovary was significantly reduced by injection of MIH, and was induced after eyestalk ablation, suggesting that the gene plays an important role in ovary development in the black tiger shrimp. Our results provide insight into the function of Pmcyclin E in the regulation of ovarian development in the black tiger shrimp.