罗氏沼虾幼体精氨酸激酶基因的cDNA克隆及其在双顺反子病毒感染后的表达特征

ARGININE KINASE GENE OF MACROBRACHIUM ROSENBERGII LARVAE CDNA CLONING AND ITS CHARACTERISTIC EXPRESSION RESEARCH AFTER DICISTRO VIRUS INFECTION

为探求罗氏沼虾精氨酸激酶(Macrobrechium rosenbergii arginine kinase,Mr AK)基因特征和与病毒感染的相关性,研究通过AK基因m RNA全长克隆,并采用QPCR检测病毒感染前后不同时间点罗氏沼虾幼体AK基因的转录差异。最终克隆得到的AK序列全长为1740 bp(Gen Bank:KT970484),其开放阅读框包含1068个碱基,编码356个氨基酸;同源性分析显示Mr AK基因编码区蛋白与多齿新米虾、南极磷虾、鲑鱼海虱和安氏伪镖水蚤的同源性分别为97%、82%、83%和79%;系统进化树分析表明,该基因与多齿新米虾的AK聚在同一分支簇,而蟹、对虾和螯虾聚在另一分支;氨基酸结构分析表明,其存在一个可能的ATP:胍基磷酸转移酶的活性位点(Cys^(286)-Pro^(287)-Thr^(288)-Asn^(289)-Leu^(290)-Gly^(291)-Thr^(292));病毒感染罗氏沼虾幼体后,其AK基因表达在第9h开始出现显著性上调,在12h时达到峰值,随后开始降低。以上研究结果表明,AK基因在无脊椎甲壳动物中存在多样性,其蛋白结构存在保守性,并且其在罗氏沼虾病毒感染过程中起到潜在的能量供给调节作用。

The current study reported the identification and characterization of arginine kinase （AK） of shrimp Macrobrechium rosenbergii （denoted as MrAK）. The full length cDNA （1740 bp） of MrAK was identified by SSH cDNA library construction and RACE approaches. Its open reading frame （ORF） was 1068 bp encoding a polypeptide of 355 amino acids. The deduced amino acid of MrAK had a potential ATP：guanidine phosphotransferases active site：（Cys^286-Pro^287-Thr^288-Asn^289-Leu^290-Gly^291-Thr^292）. Homology analysis showed that the putative MrAK shared 97%, 82%, 83% and 79% identities with AKs from Neocaridina denticulata, Euphausia superba, Lepeophtheirus salmonis, and Pseudodiaptomus annandalei, respectively. The phylogenetic tree analysis indicated that MrAK was similar to invertebrate AK, suggesting it has high evolutional conservation. Moreover, the expression of MrAK significantly up-regulated at 9h post-MrDV （M. rosenbergii dicistrovirus） immune challenge and reached peak level at 12h post-challenge. These findings suggest that MrAK might involve in host defense system against MrDV infection.