水产科学
水產科學
수산과학
FISHERIES SCIENCE
2014年
5期
288-295
,共8页
高杉%董颖%王摆%杨爱馥%陈仲%关晓燕%蒋经伟%姜北%孙红娟%周遵春
高杉%董穎%王襬%楊愛馥%陳仲%關曉燕%蔣經偉%薑北%孫紅娟%週遵春
고삼%동영%왕파%양애복%진중%관효연%장경위%강북%손홍연%주준춘
仿刺参%铜锌超氧化物歧化酶%基因表达%细菌脂多糖刺激
倣刺參%銅鋅超氧化物歧化酶%基因錶達%細菌脂多糖刺激
방자삼%동자초양화물기화매%기인표체%세균지다당자격
sea cucumber ( Apostichopus japonicus )%Cu/Zn-SOD%gene expression%bacterial LPS challenge
采用cDNA末端快速克隆技术首次克隆了仿刺参铜锌超氧化物歧化酶基因的全长cDNA序列,该基因cDNA全长1500 bp ,其中包含5′-非翻译区长129 bp ,3′-UTR长912 bp ,开放阅读框459 bp ,编码152个氨基酸,预测蛋白分子量为15.47 ku。仿刺参铜锌超氧化物歧化酶基因 cDNA推导的氨基酸序列具有保守的铜锌超氧化物歧化酶蛋白家族标签序列42 GFHIHQFGDNT52及136 GNAGGRAACGVI147,4个Cu2+结合位点(His-44,-46,-61,-118)和4个Zn2+结合位点(His-61,-69,-78,Asp-81),一对二硫键(Cys-55,-144)。氨基酸序列比对结果显示,仿刺参铜锌超氧化物歧化酶与匙胸瘿蜂和烟粉虱相似度最高,为71%。系统进化分析显示仿刺参处于无脊椎动物分支中,与紫海胆位于同一小支上。实时定量 PCR结果显示,铜锌超氧化物歧化酶基因 mRNA在仿刺参肠、体壁、肌肉、呼吸树、体腔细胞和管足中均有表达,在管足中表达量最高。在细菌脂多糖刺激后4~12 h ,体腔细胞中铜锌超氧化物歧化酶基因 mRNA表达量显著下降,表明仿刺参的铜锌超氧化物歧化酶基因对细菌脂多糖刺激有免疫应答。
採用cDNA末耑快速剋隆技術首次剋隆瞭倣刺參銅鋅超氧化物歧化酶基因的全長cDNA序列,該基因cDNA全長1500 bp ,其中包含5′-非翻譯區長129 bp ,3′-UTR長912 bp ,開放閱讀框459 bp ,編碼152箇氨基痠,預測蛋白分子量為15.47 ku。倣刺參銅鋅超氧化物歧化酶基因 cDNA推導的氨基痠序列具有保守的銅鋅超氧化物歧化酶蛋白傢族標籤序列42 GFHIHQFGDNT52及136 GNAGGRAACGVI147,4箇Cu2+結閤位點(His-44,-46,-61,-118)和4箇Zn2+結閤位點(His-61,-69,-78,Asp-81),一對二硫鍵(Cys-55,-144)。氨基痠序列比對結果顯示,倣刺參銅鋅超氧化物歧化酶與匙胸癭蜂和煙粉虱相似度最高,為71%。繫統進化分析顯示倣刺參處于無脊椎動物分支中,與紫海膽位于同一小支上。實時定量 PCR結果顯示,銅鋅超氧化物歧化酶基因 mRNA在倣刺參腸、體壁、肌肉、呼吸樹、體腔細胞和管足中均有錶達,在管足中錶達量最高。在細菌脂多糖刺激後4~12 h ,體腔細胞中銅鋅超氧化物歧化酶基因 mRNA錶達量顯著下降,錶明倣刺參的銅鋅超氧化物歧化酶基因對細菌脂多糖刺激有免疫應答。
채용cDNA말단쾌속극륭기술수차극륭료방자삼동자초양화물기화매기인적전장cDNA서렬,해기인cDNA전장1500 bp ,기중포함5′-비번역구장129 bp ,3′-UTR장912 bp ,개방열독광459 bp ,편마152개안기산,예측단백분자량위15.47 ku。방자삼동자초양화물기화매기인 cDNA추도적안기산서렬구유보수적동자초양화물기화매단백가족표첨서렬42 GFHIHQFGDNT52급136 GNAGGRAACGVI147,4개Cu2+결합위점(His-44,-46,-61,-118)화4개Zn2+결합위점(His-61,-69,-78,Asp-81),일대이류건(Cys-55,-144)。안기산서렬비대결과현시,방자삼동자초양화물기화매여시흉영봉화연분슬상사도최고,위71%。계통진화분석현시방자삼처우무척추동물분지중,여자해담위우동일소지상。실시정량 PCR결과현시,동자초양화물기화매기인 mRNA재방자삼장、체벽、기육、호흡수、체강세포화관족중균유표체,재관족중표체량최고。재세균지다당자격후4~12 h ,체강세포중동자초양화물기화매기인 mRNA표체량현저하강,표명방자삼적동자초양화물기화매기인대세균지다당자격유면역응답。
In this study ,the full‐length cDNA of Cu/Zn‐SOD gene as an immune‐related gene was cloned and characterized in sea cucumber (A postichopus japonicus) .It was found that the Cu/Zn‐SOD gene had full‐length of 1 500 bp including a 129 bp 5’‐untranslated region (UTR) ,459 bp ORF encoding 152 amino acids ,and a 912 bp 3’‐UTR with predicted molecular weight of 17 .47 ku .Four copper binding sites (His‐44 ,‐46 ,‐61 ,and‐118) and four Zn binding sites (His‐61 ,‐69 ,‐78 ,and Asp‐81) ,a conserved disulfide bond (Cys‐55 ,‐144 ) , and two conserved Cu/Zn‐SOD signatures were identified in the deduced amino acid sequence in the Cu/Zn‐SOD .The deduced amino acid sequence showed 71% amino acid sequence identity to the Cu/Zn‐SOD in Leptopilinaboulardi and Bemisia tabaci .Phylogenetic analysis revealed that the Cu/Zn‐SOD gene in the sea cucumber was close to that in purple sea urchin Strongylocentrotus purpuratus in invertebrate cluster .Quantitative real‐time PCR analysis showed that the Cu/Zn‐SOD gene was expressed in all the tested tissues including body wall ,feet ,coelomocytes ,respiratory tree ,muscle ,and intestine , the maximal expression level in feet . The expression of the Cu/Zn‐SOD mRNA was down‐regulated significantly in the coelomocytes 4—12 h after bacterial lipopolysaccharide (LPS) challenge ,indicating that the Cu/Zn‐SOD gene would be one of the immune‐related factor .