凡纳滨对虾WDS基因克隆及其表达分析
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摘要
【目的】分析WDS基因在凡纳滨对虾(Litopenaeus vannamei)各组织中的表达情况及应对不同病原感染时的表达变化,以揭示WDS在动物体除生殖外其他生理活动中的功能作用。【方法】采用RACE-PCR克隆凡纳滨对虾WDS基因(LvWDS)cDNA序列,利用在线生物信息学软件进行序列分析,并以实时荧光定量PCR分析LvWDS基因在凡纳滨对虾不同组织中的表达情况及不同病原感染后的表达变化。【结果】LvWDS基因(GenBank登录号MH330316)c DNA序列全长1880 bp,其中,开放阅读框(ORF)978 bp,5'端非编码区(5'UTR)183 bp,3'端非编码区(3'UTR)719 bp,编码325个氨基酸。LvWDS氨基酸序列含有7个WD40重复序列(保守结构域),与拟穴青蟹(Scylla paramamosain)WDS氨基酸序列的相似性最高,为99%,基于WDS氨基酸序列相似性构建的系统发育进化树也显示LvWDS与拟穴青蟹WDS的亲缘关系最近。LvWDS基因在凡纳滨对虾精囊中的相对表达量最高,在血细胞、鳃和肠道等免疫组织中处于较高表达水平,在肌肉组织中的表达水平最低。经白斑综合症病毒(WSSV)和副溶血弧菌(Vibrio parahaemolyticus)感染后,凡纳滨对虾血细胞、鳃和肠道组织中LvWDS基因的相对表达量均不同程度发生变化。【结论】WDS进化十分保守,其在不同物种中的功能稳定。LvWDS除了在凡纳滨对虾生殖发育中发挥重要作用外,可能还与免疫相关,在凡纳滨对虾抗病免疫中发挥作用。
【Objective】The purpose was to analyze the expression of WDS gene in tissues of Litopenaeus vannamei and variation in response to different pathogen infections,and reveal the function of will die slowly(WDS)in physiological activities except reproduction in animals.【Method】c DNA sequence of WDS gene(LvWDS)from L. vannamei was cloned by RACE-PCR. Bioinformatics analysis on the sequence was conducted,and distribution of LvWDS gene in various tissues as well as the expression variation after infection by different pathogens were studied by real-time fluorescence quantitative PCR.【Result】Full-length cDNA sequence of WDS gene from L. vannamei(Genbank accession number:MH330316)was 1880 bp with a 978 bp open reading frame(ORF)that encoded 325 amino acids. The 5' non-coding region(5'UTR)was 183 bp while the 3'UTR was 719 bp. LvWDS amino acid sequence contained seven WD40 repeats(conserved domains)and shared the highest(99%)amino acid similarity with WDS amino acid sequence from Scylla paramamosain. The phylogenetic tree based on WDS amino acid sequence similarity indicated that LvWDS had the closest relationship with WDS in S. paramamosain.The expression of LvWDS gene was the highest in seminal vesicle and the lowest in mu-scle. The expression levels in immunity tissues such as hemocyte,gill and intestine were high. Upon white spot syndrome virus(WSSV)and Vibrio parahaemolyticus infection,the expression of LvWDS in hemocyte,gill,and intestine of L. vannamei was changed at varying degrees.【Conclusion】WDS evolution is very conservative,and maintains stable functions in different species. LvWDS plays an important role in reproduction of L. vannamei,and may relate to immunity and also functions in disease resistance and immunity of L. vannamei.
【Objective】The purpose was to analyze the expression of WDS gene in tissues of Litopenaeus vannamei and variation in response to different pathogen infections,and reveal the function of will die slowly(WDS)in physiological activities except reproduction in animals.【Method】c DNA sequence of WDS gene(LvWDS)from L. vannamei was cloned by RACE-PCR. Bioinformatics analysis on the sequence was conducted,and distribution of LvWDS gene in various tissues as well as the expression variation after infection by different pathogens were studied by real-time fluorescence quantitative PCR.【Result】Full-length cDNA sequence of WDS gene from L. vannamei(Genbank accession number:MH330316)was 1880 bp with a 978 bp open reading frame(ORF)that encoded 325 amino acids. The 5' non-coding region(5'UTR)was 183 bp while the 3'UTR was 719 bp. LvWDS amino acid sequence contained seven WD40 repeats(conserved domains)and shared the highest(99%)amino acid similarity with WDS amino acid sequence from Scylla paramamosain. The phylogenetic tree based on WDS amino acid sequence similarity indicated that LvWDS had the closest relationship with WDS in S. paramamosain.The expression of LvWDS gene was the highest in seminal vesicle and the lowest in mu-scle. The expression levels in immunity tissues such as hemocyte,gill and intestine were high. Upon white spot syndrome virus(WSSV)and Vibrio parahaemolyticus infection,the expression of LvWDS in hemocyte,gill,and intestine of L. vannamei was changed at varying degrees.【Conclusion】WDS evolution is very conservative,and maintains stable functions in different species. LvWDS plays an important role in reproduction of L. vannamei,and may relate to immunity and also functions in disease resistance and immunity of L. vannamei.
引文
曹煜成,徐煜,黄小帅,胡晓娟,李卓佳,李凡,陈勇贵,文国樑.2018.铜绿微囊藻对WSSV潜伏感染对虾的致死效应[J].南方农业学报,49(8):1648-1653.[Cao Y C,Xu Y,Huang X S,Hu X J,Li Z J,Li F,Chen Y G,Wen G L.2018.Lethal effects of Microcystis aeruginosa to Litopenaeus vannamei infected by WSSV[J].Journal of Southern Agriculture,49(8):1648-1653.]
高洁.2013.拟穴青蟹性腺差异的转录组学分析[D].厦门:集美大学.[Gao J.2013.Comparative transcriptome analysis of gonad of the Scylla paramamosain[D].Xiamen:Jimei University.]
黄金凤,杨奇慧,董晓慧,迟淑艳,刘泓宇,谭北平,章双.2017.凡纳滨对虾ARMC8基因的克隆及表达[J].水产学报,41(2):171-181.[Huang J F,Yang Q H,Dong X H,Chi S Y,Liu H Y,Tan B P,Zhang S.2017.Cloning and expression profile analysis of ARMC8 gene from Litopenaeus vannamei[J].Journal of Fisheries of China,41(2):171-181.]
吕莉,朱志东,邓剑壕,冯豆,蔡延渠,朱宇嘉,朱盛山.2018.对虾弧菌病的研究进展[J].水产学杂志,31(4):53-58.[LüL,Zhu Z D,Deng J H,Feng D,Cai Y Q,Zhu Y J,Zhu S S.2018.Current research of prawn vibriosis:A review[J].Chinese Journal of Fisheries,31(4):53-58.]
明磊,戴习林,江宗冰,丁福江.2018.高通量测序在罗氏沼虾抗病力比较中的应用[J].河南农业科学,47(8):134-141.[Ming L,Dai X L,Jiang Z B,Ding F J.2018.Application of high-throughput sequencing in the comparison of disease resistance of Macrobrachium rosenbergii[J].Journal of Henan Agricultural Sciences,47(8):134-141.]
农业部渔业渔政管理局.2016.2016年中国渔业统计年鉴[M].北京:中国农业出版社.[Bureau of Fisheries of the Ministry of Agriculture.2016.2016 China Fishery Statistical Yearbook[M].Beijing:China Agriculture Press.]
钱美睿.2013.罗氏沼虾smu-1基因的筛选、表达及其功能研究[D].杭州:中国计量学院.[Qian M R.2013.Screening,expression and the functional analysis of smu-1 genes from Macrochium rosenbergii[D].Hangzhou:China Jiliang University.]
周井娟.2016.中国对虾养殖业发展轨迹及技术变迁[J].中国农学通报,32(8):22-29.[Zhou J J.2016.Development path and technological change of shrimp aquaculture in China[J].Chinese Agricultural Science Bulletin,32(8):22-29.]
Burge E J,Madigan D J,Burnett L E,Burnett K G.2007.Lysozyme gene expression by hemocytes of Pacific white shrimp,Litopenaeus vannamei,after injection with Vibrio[J].Fish&Shellfish Immunology,22(4):327-339.
Coronin P J.2008.Diversity of WD-repeat proteins[J].Subcellular Biochemistry,48:116-123.
Hollmann M,Simmerl E,Sch?fer U,Sch?fer M.2002.The essential Drosophila melanogaster gene wds(will die slowly)codes for a WD-repeat protein with seven repeats[J].Molecular Genetics and Genomics,268(4):425-433.
Lai C Y,Cheng W,Kuo C M.2005.Molecular cloning and characterisation of prophenoloxidase from haemocytes of the white shrimp,Litopenaeus vannamei[J].Fish&Shellfish Immunology,18(5):417-430.
Li D,Roberts R.2001.Human genome and diseases:?WD-repeat proteins:Structure characteristics,biological function,and their involvement in human diseases[J].Cellular and Molecular Life Sciences,58(14):2085-2097.
Li F,Xiang J.2013.Signaling pathways regulating innate immune responses in shrimp[J].Fish&Shellfish Immunology,34(4):973-980.
Li Y,Wang H,Xia R,Wu S,Shi S,Su J,Liu Y,Qin L,Wang Z.2011.Molecular cloning,expression pattern and phylogenetic analysis of the will die slowly gene from the Chinese oak silkworm,Antheraea pernyi[J].Molecular Biology Reports,38(6):3795-3803.
Lightner D V,Poulos B T,Tang-Nelson K F,Pantoja C R,Nunan L M,Navarro S A,Redman R M,Mohney L L.2005.Application of molecular diagnostic methods to penaeid shrimp diseases:Advances of the past 10 years for control of viral diseases in farmed shrimp[J].Developments in Biologicals,126:117-122.
Thitamadee S,Prachumwat A,Srisala J,Jaroenlak P,Salachan P V,Sritunyalucksana K,Flegel T,Itsathitphaisarn O.2016.Review of current disease threats for cultivated penaeid shrimp in Asia[J].Aquaculture,452:69-87.
Villanueva M A,Tania Islas-Flores,Ullah H.2016.Editorial:Signaling through WD-repeat proteins in plants[J].Frontiers in Plant Science,7:1157.doi:10.3389/fpls.2016.01157.
Xu D,Liu W,Alvarez A,Huang T.2014.Cellular immune responses against viral pathogens in shrimp[J].Developmental and Comparative Immunology,47(2):287-297.
Ye L L,Zhang X Y,Xiong X Q,Xing T,Wang X J,Lu J,Sun S L.2014.Expression pattern analysis of will die slowly genes in Arabidopsis[J].Russian Journal of Plant Physiology,61(6):873-879.
Zanjani N T,Miranda-Saksena M,Cunningham A L,Dehghani F.2018.Antimicrobial peptides of marine crustaceans:The potential and challenges of developing therapeutic agents[J].Current Medicinal Chemistry,25(19):2245-2259.
Zhang S,Li C Z,Yang Q H,Dong X H,Chi S Y,Liu H Y,Shi L L,Tan B P.2016.Molecular cloning,characterization and expression analysis of Wnt4,Wnt5,Wnt6,Wnt7,Wnt10 and Wnt16 from Litopenaeus vannamei[J].Fish&Shellfish Immunology,54:445-455.
Zhao Z Y,Yin Z X,Xu X P,Weng S P,Rao X Y,Dai Z X,Luo Y W,Yang G,Li Z S,Guan H J,Li S D,Chan S M,Yu X Q,He J G.2009.A novel C-type lectin from the shrimp Litopenaeus vannamei possesses anti-white spot syndrome virus activity[J].Journal of Virology,83(1):347-56.
高洁.2013.拟穴青蟹性腺差异的转录组学分析[D].厦门:集美大学.[Gao J.2013.Comparative transcriptome analysis of gonad of the Scylla paramamosain[D].Xiamen:Jimei University.]
黄金凤,杨奇慧,董晓慧,迟淑艳,刘泓宇,谭北平,章双.2017.凡纳滨对虾ARMC8基因的克隆及表达[J].水产学报,41(2):171-181.[Huang J F,Yang Q H,Dong X H,Chi S Y,Liu H Y,Tan B P,Zhang S.2017.Cloning and expression profile analysis of ARMC8 gene from Litopenaeus vannamei[J].Journal of Fisheries of China,41(2):171-181.]
吕莉,朱志东,邓剑壕,冯豆,蔡延渠,朱宇嘉,朱盛山.2018.对虾弧菌病的研究进展[J].水产学杂志,31(4):53-58.[LüL,Zhu Z D,Deng J H,Feng D,Cai Y Q,Zhu Y J,Zhu S S.2018.Current research of prawn vibriosis:A review[J].Chinese Journal of Fisheries,31(4):53-58.]
明磊,戴习林,江宗冰,丁福江.2018.高通量测序在罗氏沼虾抗病力比较中的应用[J].河南农业科学,47(8):134-141.[Ming L,Dai X L,Jiang Z B,Ding F J.2018.Application of high-throughput sequencing in the comparison of disease resistance of Macrobrachium rosenbergii[J].Journal of Henan Agricultural Sciences,47(8):134-141.]
农业部渔业渔政管理局.2016.2016年中国渔业统计年鉴[M].北京:中国农业出版社.[Bureau of Fisheries of the Ministry of Agriculture.2016.2016 China Fishery Statistical Yearbook[M].Beijing:China Agriculture Press.]
钱美睿.2013.罗氏沼虾smu-1基因的筛选、表达及其功能研究[D].杭州:中国计量学院.[Qian M R.2013.Screening,expression and the functional analysis of smu-1 genes from Macrochium rosenbergii[D].Hangzhou:China Jiliang University.]
周井娟.2016.中国对虾养殖业发展轨迹及技术变迁[J].中国农学通报,32(8):22-29.[Zhou J J.2016.Development path and technological change of shrimp aquaculture in China[J].Chinese Agricultural Science Bulletin,32(8):22-29.]
Burge E J,Madigan D J,Burnett L E,Burnett K G.2007.Lysozyme gene expression by hemocytes of Pacific white shrimp,Litopenaeus vannamei,after injection with Vibrio[J].Fish&Shellfish Immunology,22(4):327-339.
Coronin P J.2008.Diversity of WD-repeat proteins[J].Subcellular Biochemistry,48:116-123.
Hollmann M,Simmerl E,Sch?fer U,Sch?fer M.2002.The essential Drosophila melanogaster gene wds(will die slowly)codes for a WD-repeat protein with seven repeats[J].Molecular Genetics and Genomics,268(4):425-433.
Lai C Y,Cheng W,Kuo C M.2005.Molecular cloning and characterisation of prophenoloxidase from haemocytes of the white shrimp,Litopenaeus vannamei[J].Fish&Shellfish Immunology,18(5):417-430.
Li D,Roberts R.2001.Human genome and diseases:?WD-repeat proteins:Structure characteristics,biological function,and their involvement in human diseases[J].Cellular and Molecular Life Sciences,58(14):2085-2097.
Li F,Xiang J.2013.Signaling pathways regulating innate immune responses in shrimp[J].Fish&Shellfish Immunology,34(4):973-980.
Li Y,Wang H,Xia R,Wu S,Shi S,Su J,Liu Y,Qin L,Wang Z.2011.Molecular cloning,expression pattern and phylogenetic analysis of the will die slowly gene from the Chinese oak silkworm,Antheraea pernyi[J].Molecular Biology Reports,38(6):3795-3803.
Lightner D V,Poulos B T,Tang-Nelson K F,Pantoja C R,Nunan L M,Navarro S A,Redman R M,Mohney L L.2005.Application of molecular diagnostic methods to penaeid shrimp diseases:Advances of the past 10 years for control of viral diseases in farmed shrimp[J].Developments in Biologicals,126:117-122.
Thitamadee S,Prachumwat A,Srisala J,Jaroenlak P,Salachan P V,Sritunyalucksana K,Flegel T,Itsathitphaisarn O.2016.Review of current disease threats for cultivated penaeid shrimp in Asia[J].Aquaculture,452:69-87.
Villanueva M A,Tania Islas-Flores,Ullah H.2016.Editorial:Signaling through WD-repeat proteins in plants[J].Frontiers in Plant Science,7:1157.doi:10.3389/fpls.2016.01157.
Xu D,Liu W,Alvarez A,Huang T.2014.Cellular immune responses against viral pathogens in shrimp[J].Developmental and Comparative Immunology,47(2):287-297.
Ye L L,Zhang X Y,Xiong X Q,Xing T,Wang X J,Lu J,Sun S L.2014.Expression pattern analysis of will die slowly genes in Arabidopsis[J].Russian Journal of Plant Physiology,61(6):873-879.
Zanjani N T,Miranda-Saksena M,Cunningham A L,Dehghani F.2018.Antimicrobial peptides of marine crustaceans:The potential and challenges of developing therapeutic agents[J].Current Medicinal Chemistry,25(19):2245-2259.
Zhang S,Li C Z,Yang Q H,Dong X H,Chi S Y,Liu H Y,Shi L L,Tan B P.2016.Molecular cloning,characterization and expression analysis of Wnt4,Wnt5,Wnt6,Wnt7,Wnt10 and Wnt16 from Litopenaeus vannamei[J].Fish&Shellfish Immunology,54:445-455.
Zhao Z Y,Yin Z X,Xu X P,Weng S P,Rao X Y,Dai Z X,Luo Y W,Yang G,Li Z S,Guan H J,Li S D,Chan S M,Yu X Q,He J G.2009.A novel C-type lectin from the shrimp Litopenaeus vannamei possesses anti-white spot syndrome virus activity[J].Journal of Virology,83(1):347-56.
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