管氏肿腿蜂毒液精氨酸激酶基因的克隆与表达分析

Molecular cloning and expression analysis of arginine kinase from Scleroderma guani

为了解精氨酸激酶作为寄生蜂毒液蛋白的功能,本研究克隆了管氏肿腿蜂毒液精氨酸激酶基因的开放阅读框,分析其表达特征,并测定了该毒液蛋白在毒液中的酶活性。克隆获得的毒液精氨酸激酶基因的开放阅读框长1 068 bp,编码了355个氨基酸,其理论分子量为39.71 kDa,等电点为5.86,并具有精氨酸激酶典型的N端和C端结构域,以及精氨酸和ADP结合位点、ATP-胍基磷酸转移酶活性位点和高度保守的天冬氨酸和精氨酸残基。系统进化分析表明,膜翅目精氨酸激酶分为两个亚家族,管氏肿腿蜂精氨酸激酶属于亚家族1。荧光定量PCR分析结果显示,该毒液蛋白基因在卵、幼虫、蛹、雌成虫中的表达量逐渐上升,至羽化10 d时达最高。在不同组织中,该基因在头部和毒液器官中的表达量较高。通过酶活测定发现,管氏肿腿蜂毒液中精氨酸激酶的酶活性为5.18 U/g蛋白。该研究有助于今后揭示寄生蜂毒液精氨酸激酶的功能与作用机制。

In order to further understand the role of arginine kinase in the parasitoid venoms, we cloned the open reading frame(ORF) sequence of the venom arginine kinase gene from Scleroderma guani followed by the analyses of its expression and enzymatic activity. The length of cloned ORF of venom arginine kinase gene of S.guani is 1 068 bp, encoding 355 amino acid with the predicted molecular weight of 39.71 kDa and the isoelectric point of 5.86. It also has typical signatures of arginine kinase, including N-terminal and C-terminal domains, arginine and ADP binding sites, ATP-guanido phosphotransferase active sites, and highly conserved aspartate and arginine residues. Phylogenetic analysis indicated that Hymenoptera arginine kinases were divided into two groups, the arginine kinases of S.guani clustered in group 1. Fluorescent quantitative real time PCR analysis revealed that its gene expression was gradually increased in eggs, larvae, pupa, and female adults with the highest level detected at 10 days after emergence. This gene showed higher expression in head and venom apparatus than in other tissues. By enzymatic assay, it was found that 1 g venom has the arginine kinase activity of 5.18 U. These results are helpful for investigating the function and acton mechanism of venom arginine kinase in parasitoid wasps.