红螯光壳螯虾(Cherax quadricarinatus)酚氧化酶原基因的克隆与表达分析

cDNA cloning and expression analysis of prophenoloxidasein Cherax quadricarinatus

摘要:

利用RACE技术从红螯光壳螯虾血细胞中克隆到酚氧化酶原基因CqproPO,CqproPO基因cDNA全长为2962bp,开放阅读框为1998bp,编码665个氨基酸,N端含有38个氨基酸的信号肽序列,其结构中含有两个铜离子结合位点,预测分子量为75.86KDa；同源性比对结果显示红螯光壳螯虾CqproPO与克氏原螯虾酚氧化酶原的同源性最高为79%,其次是淡水螯虾74%、挪威龙虾69%、美国龙虾67%等；进化分析发现CqproPO与克氏原鳌虾、淡水螯虾、挪威龙虾、美国龙虾等虾的酚氧化酶原亲缘关系最近；Realtime-PCR实验结果表明,CqproPO 在血细胞中表达水平最高,其次是肠、触角腺、鳃等；在肝胰腺中有适量表达；WSSV感染后红螯光壳螯虾CqproPO mRNA在血细胞、肝胰腺和鰓组织中具有不同的时空表达趋势,但感染组和免疫后感染组mRNA表达量分别在感染后12h和24h达到最大值,且在3种组织中2个感染组的CqproPO表达量约为对照组的1.3-2.55倍,显著高于对照组(P<0.05),之后CqproPO基因的转录水平明显下降。免疫后再受病毒感染的虾,CqproPO mRNA的表达量在3种组织中总体高于感染组,表明免疫增强剂可使机体的抗病毒能力增强,对防御WSSV感染具有一定的免疫保护作用。

Abstract:

The Australian redclaw crayfish, Cherax quadricarinatus, is a crustacean belonging to the order Decapoda, family Parastacidae. It is currently one of the most important commercially farmed fresh crayfish in the world due to its many advantages, including polyphagia, fast growth and easy culture, as well as being rich in protein and low in cholesterol. During the last two decades, the environmental and health problems in redclaw crayfish culture have resulted in the outbreak of infectious diseases with the constantly expanding scale of aquaculture and the rapid development of intensive culture techniques of C. quadricarinatus in China.White spot syndrome virus (WSSV) as the pathogenic agent caused acute and lethal infect ion of shrimp, resulting in mass economic losses to shrimp aquaculture industry all around the world especially in China. In recent years, the cultivation of redclaw crayfish (Cherax quadricarinatus) is developing, and the disease of C. quadricarinatus was one of the major factors in its culture and even caused redclaw crayfish to die. The viral disease was found in polyculture of redclaw crayfish with Penaeus vannamei. To explore the pathogenic mechanism of C. quadricarinatus infected by WSSV (white spot syndrome virus) , a prophenoloxidase gene（CqproPO） was cloned from haemocytes of C. quadricarinatus by Rapid Amplification Complementary DNA Ends(RACE) method, the proPO gene expression patterns in different tissues and the mRNA expression of proPO gene in hemocyte, hepatopancreas and gill tissues of C. quadricarinatus infected by WSSV artificially were studied. The results indicated that the full length cDNA of CqproPO consisted of 2962bp with a 1998 bp Open Reading Frame(ORF)，which encoded 665 amino acids，and a predicted signal peptide of 38 amino acids, and the predicted molecular mass was 75.86KDa. Sequence analysis showed CqproPO contained two conserved copperbinding sites; The secondary and tertiary structure assay also showed that the CqproPO has α-helices and β-strands, which is delimiting a cavity where the hydrophobic ligands are bound just as other HCs. ORF contains two tyrosine kinase phosphorylation sites, 13 casein kinase II phosphorylation sites, 7 protein kinase C phosphorylation sites, one dependent on cAMP-and cGMP protein kinase phosphorylation sites and three N-glycosylation sites, which these sites were structural basis of the physiological functions completed. The deduced amino acids sequence of CqproPO sharing 79% homology with Procambarus clarkii and 74%, 69%, 67%, 67% with Pacifastacus leniusculus, Nephrops norvegicus, Homarus americanus, Homarus gammarus respectively; Phylogenetic analysis revealed that CqproPO and prophenoloxidase from P. clarkii, P. leniusculus, N.norvegicus, H.americanus, H. gammarus and Panulirus longipes were in the same phylogenetic branches; The Realtime-PCR results showed that CqproPO was widely distributed, with the highest expression level in haemocytes, small amount of expression in intestine, antennal gland, gills, ovary and hepatopancreas, detectable expression level in stomach and muscle, while expression was almost undetectable in testis; The expression level of prophenoloxidase (proPO) in haemocytes, hepatopancreas and gills from C. quadricarinatus were studied and compared by means of artificial WSSV infection. The results indicated that the expression level of CqproPO in the non-immunized infected group (Group II) and immunized infected group (Group III) reached the maximum at 12 h and 24h, which was 1.3-2.55 times higher than that in the control group, and was noticeable higher than the controls (p <0.05). But the expression level of prophenoloxidase gene had sharply declined with the time extending of the infection. The expression level of proPO gene from crayfishes injected by immune polysaccharides before an infected virus (GroupⅢ) were higher than the directly affected groups (GroupⅡ) in haemocytes, hepatopancreas and gills. These observations indicated that the immunopotentiator could improve the innate anti-viral ability of the crustaceans against WSSV.