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菲律宾蛤仔硒依赖性谷胱甘肽过氧化物酶基因克隆、表达及其对脂多糖和肽聚糖的刺激响应

Molecular characterization,expression and immune responses to lipopolysaccharide and peptidoglycan stimulation of Se-GPx gene in Manila clam Ruditapes philippinarum
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摘要 为探究菲律宾蛤仔Ruditapes philippinarum硒依赖性谷胱甘肽过氧化物酶(Se-GPx)基因的序列特征、组织表达模式及其在不同壳色蛤仔免疫应答中的作用,利用RACE方法获得分析菲律宾蛤仔两个Se-GPx基因(Se-GPx-a和Se-GPx-b)全长序列,采用RT-qRCR方法分析基因表达规律。结果表明:RpSe-GPx-a和RpSe-GPx-b基因开放阅读框(ORF)长度分别为582 bp和633 bp,分别编码193和210个氨基酸,在3′非翻译区(UTR)均含有保守硒代半胱氨酸插入序列(SECIS)元件;氨基酸序列分析发现,RpSe-GPx-a和RpSe-GPx-b序列中含有与酶结构和功能至关重要的特征,包括终止密码子UGA编码的硒代半胱氨酸,GPx标签序列(GKVVLVENVASLUGTT)和活性位点序列(WNFEKF)均高度保守;系统进化分析发现,RpSe-GPx与其他软体动物物种具有较近的亲缘关系;组织表达分析发现,RpSe-GPx在肝胰腺中表达量最高(P<0.05),在外套膜、鳃组织中表达量次之,在闭壳肌中表达量最低(P<0.05);用脂多糖(LPS)和肽聚糖(PGN)分别注射3种壳色蛤仔(白蛤、白斑马、橙蛤),LPS刺激后,白蛤Se-GPx-a和Se-GPx-b基因表达量分别在12、6 h时达到最大值(P<0.05),白斑马蛤Se-GPx-a和Se-GPx-b基因表达量分别在48、6 h时达到最大值(P<0.05),橙蛤Se-GPx-a和Se-GPx-b基因表达量分别在3、24 h时达到最大值(P<0.05);PGN刺激后,白蛤、白斑马蛤和橙蛤Se-GPx-a和Se-GPx-b基因表达量分别在24、24、48 h时达到最大值(P<0.05)。研究表明,3种壳色蛤仔黑色素形成过程的差异可能是导致其GPx基因对LPS和PGN刺激表现出不同响应的重要原因之一。 The selenium-dependent glutathione peroxidase(Se-GPx)gene(designated as Se-GPx-a and Se-GPx-b was cloned and expressed in adductor muscle,mantle,gill,water tube,gonad,hepatopancreas,and feet of orange,white and zebrawhite Manila clam Ruditapes philippinarum with shell length of(2.0±0.5)cm injected hemolymphically with lipopolysaccharide solution(LPS)and peptidoglycan solution(PGN)of 100μg/mL at a dose of 50μL per clam at water temperature of(18.0±0.5)℃by RACE technology and RT-PCR method to investigate the molecular characterization,expression and immune responses to lipopolysaccharide and peptidoglycan stimulation of selenium-dependent Se-GPx gene in Manila clam.It was found that the full-length cDNA of Se-GPx-a and Se-GPx-b had the open reading frame(ORF)of 582 bp in the RpSe-GPx-a and 633 bp in RpSe-GPx-b,encoding two polypeptides of 193 and 210 amino acids in length.There were conserved selenocysteine insertion sequence(SECIS)in the 3′untranslated region(UTR)of the RpSe-GPx showing all the conserved features critical for the fundamental structure and function of glutathione peroxidase,including the selenocysteine encoded by stop codon UGA,the GPx signature motif(GKVVLVENVASLUGTT)and the active site motif(WNFEKF),with high homology of RpSe-GPx with other mollusc species.Tissue expression analysis revealed that there was the maximal expression level of RpSe-GPx in the hepatopancreas(P<0.05),followed by the mantle and gills,and the minimal expression level in the adductor muscle(P<0.05).The maximal expression level of Se-GPx-a and Se-GPx-b mRNA in Manila clam injected hemolymphically with LPS were detected in the white-shell color group at 12 and 6 h,and in the white zebra-shell color group at 48 h and 6 h,and in the orange-shell color group at 3 h and 24 h(P<0.05).The maximal expression level of Se-GPx-a and Se-GPx-b in Manila clam injected hemolymphically with PGN were all significantly induced at 24 h in white-shell color group,and at 24 h in the white zebra-shell color group,and at 48 h in the orange-shell color group(P<0.05).The findings indicated that different responses of the RpSe-GPx gene to LPS and PGN stimulation were attributed in the difference in the melanin formation process in the three shell-color clam,which provided data for shell color selection breeding and healthy farming of Manila clam.
作者 王珺 梁腾 王化敏 闫喜武 丁鉴锋 WANG Jun;LIANG Teng;WANG Huamin;YAN Xiwu;DING Jianfeng(College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China;Engineering Research Center of Shellfish Culture and Breeding in Liaoning Province, Dalian 116023, China)
出处 《大连海洋大学学报》 CAS CSCD 北大核心 2021年第1期30-37,共8页 Journal of Dalian Ocean University
基金 国家重点研发计划项目(2018YFD0901404)。
关键词 菲律宾蛤仔 Se-GPx 克隆 基因表达 Ruditapes philippinarum Se-GPx clone gene expression
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