斑节对虾CDK2基因全长cDNA克隆及表达分析

Molecular cloning and expression analysis of CDK2 gene from black tiger shrimps( Penaeus monodon)

该研究以斑节对虾(Penaeus monodon)c DNA文库中的CDK2片段为基础,利用RACE技术获得Pm CDK2的c DNA全长,并利用生物信息学对其进行了分析。Pm CDK2基因全长1 679 bp,包括编码306个氨基酸的921 bp的开放阅读框(ORF),258 bp的5'UTR和500 bp的3'UTR。同源性分析显示,Pm CDK2与罗氏沼虾(Macrobrachium rosenbergii)等物种的CDK2具有较高的同源性。使用荧光定量PCR技术研究了Pm CDK2的mRNA在不同组织和卵巢发育各时期的相对表达量和变化模式,结果显示,Pm CDK2在斑节对虾的脑、心、淋巴、肝胰腺、性腺等组织中均有表达,其中精巢中表达量显著高于其他组织;卵巢发育阶段表达分析则显示Pm CDK2在Ⅲ期表达量最高,其次是Ⅳ期。通过原核表达技术成功获得了含有6个His标签的融合蛋白,并对融合蛋白进行Western Blot试验,初步探讨了重组蛋白表达情况。结果表明,Pm CDK2基因在斑节对虾卵巢发育过程中可能发挥了重要作用,该结果对进一步探究斑节对虾卵巢发育机理提供了一定的理论依据。

The full-length c DNA sequence of CDK2 from black tiger shrimps（ Penaeus monodon）（ denoted as Pm CDK2） was obtained by high throughput transcriptome sequencing and RACE. The c DNA of Pm CDK2 was 1 679 bp,including an open reading frame（ ORF）of 921 bp encoding a polypeptide of 306 amino acids,a 5＇UTR of 258 bp and a 3＇UTR of 500 bp. Blast and phylogenetic analyses suggest that Pm CDK2 is a new member of the shrimp CDKs family. Pm CDK2 mRNA expression was detected in seven tissues by real-time PCR.Expression was the highest in the testis and moderate in the ovary,lymph,muscle and so on. While at different stages of ovary development,Pm CDK2 expressed most at Stage Ⅲ,indicating that Pm CDK2 might play an important role in ovary development for black tigershrimp. The results provide insight into the function of Pm CDK2 in regulation of ovarian development of black tiger shrimp.