粘虫过氧化物酶MsPOD基因的克隆及不同温度胁迫的诱导效应
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摘要
过氧化物酶(POD)是昆虫体内一种很关键的抗氧化酶,在维持昆虫体内氧化还原的动态平衡及保护昆虫免受氧化损伤方面起到重要的作用。本试验通过转录组测序方法获得一条粘虫过氧化物酶基因的cDNA序列,该序列命名为MsPOD(GenBank登录号:MH606240)。该序列全长2433 bp,开放阅读框长度为2061 bp,编码686个氨基酸组成的多肽,分子量约76.1 ku,等电点5.68,具有1个标志性的动物亚铁血红素过氧化物酶细胞粘附蛋白结构域。氨基酸序列比对表明,MsPOD的氨基酸序列与鳞翅目夜蛾科其他昆虫亲缘关系较近,其中与棉铃虫POD氨基酸序列同源性最高,达92%。基因表达水平研究发现,MsPOD基因在不同发育阶段和不同组织中差异表达,在蛹期和唾腺中表达量最高。温度梯度诱导后,MsPOD基因在不同时间点的表达量具有显著差异,经35℃下12h处理后表达量最高。研究结果为进一步研究过氧化物酶在昆虫体内的保护性作用以及利用该基因进行分子设计来防治粘虫奠定理论基础。
Peroxidase is a key antioxidant enzyme in insects and plays an important role in maintaining redox homeostasis in insects and protecting insects from oxidative damage. In this study, a novel peroxidase cDNA sequence MsPOD(GenBank accession: No. MH606240) was obtained from Mythimna separata by transcriptome sequencing technology. The full-length cDNA sequence MsPOD was 2433 bp, with ORF length of 2061 bp, encoding a polypeptide consisting of 686 amino acids. The peptide was characterized by a molecular mass of about 76.1 ku and an isoelectric point of about 5.68, and distinct domain of animal heme peroxidase cell adhesion protein. Amino acids alignment analysis showed that MsPOD amino acid sequence was close in homology to that of other Noctuidae insects, with the highest homology of 92% with that in Helicoverpa armigera. MsPOD expressed specifically at different developmental stages and in different tissues, the highest expression occurring at pupal stage and in salivary glands. Expression of MsPOD also varied with different temperatures, reaching the highest level at 12 h post treatment at 35℃. These results lay a foundation for the further study of protective effects of MsPOD and utilizing it to design molecular method to control M. separata.
Peroxidase is a key antioxidant enzyme in insects and plays an important role in maintaining redox homeostasis in insects and protecting insects from oxidative damage. In this study, a novel peroxidase cDNA sequence MsPOD(GenBank accession: No. MH606240) was obtained from Mythimna separata by transcriptome sequencing technology. The full-length cDNA sequence MsPOD was 2433 bp, with ORF length of 2061 bp, encoding a polypeptide consisting of 686 amino acids. The peptide was characterized by a molecular mass of about 76.1 ku and an isoelectric point of about 5.68, and distinct domain of animal heme peroxidase cell adhesion protein. Amino acids alignment analysis showed that MsPOD amino acid sequence was close in homology to that of other Noctuidae insects, with the highest homology of 92% with that in Helicoverpa armigera. MsPOD expressed specifically at different developmental stages and in different tissues, the highest expression occurring at pupal stage and in salivary glands. Expression of MsPOD also varied with different temperatures, reaching the highest level at 12 h post treatment at 35℃. These results lay a foundation for the further study of protective effects of MsPOD and utilizing it to design molecular method to control M. separata.
引文
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[23]王玲,江幸福,罗礼智,等.粘虫类钙粘蛋白基因的克隆、序列分析及时空表达[J].昆虫学报,2011,54(10):1094-1103.
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[25]Zhao X,Smartt C T,Li J,et al.Aedes aegypti peroxidase gene characterization and developmental expression[J].Insect Biochemistry and Molecular Biology,2001,31(4/5):481-490.
[26]Lushchak V I.Environmentally induced oxidative stress in aquatic animals[J].Aquatic Toxicology,2011,101(1):13-30.
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[28]Corona M,Robinson G E.Genes of the antioxidant system of the honey bee:Annotation and Phylogeny[J].Insect Molecular Biology,2006,15(5):687-701.
[29]Kobayashi-Miura M,Shioji K,Hoshino Y,et al.Oxygen sensing and redox signaling:the role of thioredoxin in embryonic development and cardiac diseases[J].American Journal of Physiology Heart&Circulatory Physiology,2007,292(5):2040-2050.
[30]李毅平,龚和.昆虫体内抗氧化系统研究进展[J].生命科学,1998(5):240-243.
[31]杨唐斌,梅尚筠.热休克对家蚕幼虫抗氧化酶活性的影响[J].生物化学与生物物理进展,1996(2):153-156.
[32]李庆,吴蕾,杨刚,等.温度和紫外辐射胁迫对西藏飞蝗抗氧化系统的影响[J].生态学报,2012,32(10):3189-3197.
[33]王艳敏,仵均祥,万方浩.昆虫对极端高低温胁迫的响应研究[J].环境昆虫学报,2010,32(2):250-255.
[34]庹瑞锐,赵运林,董萌,等.土壤重金属镉、铅污染的植物修复技术研究进展[J].贵州农业科学,2011,39(6):216-220.
[2]Jin L H,Bahn J H,Eum W S,et al.Transduction of human catalase mediated by an HIV-1 TAT protein basic domain and arginine-rich peptides into mammalian cells[J].Free Radical Biology&Medicine,2001,31(11):1509-1519.
[3]Dandona P,Mohanty P,Ghanim H,et al.The suppressive effect of dietary restriction and weight loss in the obese on the generation of reactive oxygen species by leukocytes,lipid peroxidation,and protein carbonylation[J].Journal of Clinical Endocrinology and Metabolism,2000,86(1):355-362.
[4]Wiseman H,Halliwell B.Damage to DNA by reactive oxygen and nitrogen species:role in inflammatory disease and progression to cancer[J].Biochemical Journal,1996,313:17-29.
[5]李爽,蔡梦婷,马婉颖,等.意大利蝗和西伯利亚蝗高温耐受能力及酶活性比较研究[J].应用昆虫学报,2016,53(5):1077-1083.
[6]Sritunyalucksana K,Wongsuebsantati K,Johansson M W,et al.Peroxinectin,a cell adhesive protein associated with the proPO system from the black tiger shrimp,Penaeus monodon[J].Developmental and Comparative Immunology,2001,25(5-6):353-363.
[7]ZámockyM,Hofbauer S,Schaffner I,et al.Independent evolution of four heme peroxidase superfamilies[J].Archives of Biochemistry and Biophysics,2015,574(9):108-119.
[8]Zamocky M,Jakopitsch C,Furtmüller P G,et al.The peroxidase-cyclooxygenase superfamily:Reconstructed evolution of critical enzymes of the innate immune system[J].Proteins-structure Function and Bioinformatics,2008,72(2):589-605.
[9]Johansson M W,Lind M I,Holmblad T,et al.Peroxinectin,a novel cell adhesion protein from crayfish blood[J].Biochemical and Biophysical Research Communications,1995,216(3):1079-1087.
[10]Davies M J,Hawkins C L,Pattison D I,et al.Mammalian heme peroxidases:from molecular mechanisms to health implications[J].Antioxidants and Redox Signaling,2008,10(7):1199-1234.
[11]Daiyasu H,Toh H.Molecular evolution of the myeloperoxidase family[J].Journal of Molecular Evolution,2000,51(5):433-445.
[12]申忠健,张松斗,刘彦君,等.棉铃虫过氧化物酶基因HaPOD的克隆和表达分析[J].应用昆虫学报,2016,53(5):953-961.
[13]Park J,Stanley D,Kim Y.Roles of peroxinectin in PGE2-mediated cellular immunity in Spodoptera exigua[J].PLoS ONE,2014,9(9):e105717.
[14]Vázquez M,Rodríguez R,Zurita M.A new peroxinectin-like gene preferentially expressed during oogenesis and early embryogenesis in Drosophila melanogaster[J].Development Genes and Evolution,2002,212(11):526-529.
[15]见煜坤.褐飞虱亚铁血红素过氧化物酶基因N125323生物学功能解析[D].杭州:浙江大学,2016,21-34.
[16]李品武,付雪莲,陈世春,等.温度胁迫对茶淡黄刺蛾四种保护酶活力和总抗氧化力的影响[J].应用昆虫学报,2016,53(4):809-816.
[17]Jia F X,Dou W,Hu F,et al.Effects of thermal stress on lipid peroxidation and antioxidant enzyme activities of oriental fruit fly,Bactrocera dorsalis(Diptera:Tephritidae)[J].Florida Entomologist,2011,94(4):956-963.
[18]Kim R O,Rhee J S,Won E J,et al.Ultraviolet B retards growth,induces oxidative stress,and modulates DNA repair-related gene and heat shock protein gene expression in the monogonont rotifer,Brachionus sp.[J].Aquatic Toxicology,2011,101(3/4):529-539.
[19]苏添龙,谢克强,崔汝强,等.昆虫y-box蛋白的研究进展[J].贵州农业科学,2015,43(10):55-59.
[20]杨静,Nasir Ali Baloch,樊东.黏虫热休克蛋白Hsp90基因的克隆及不同温度对其诱导反应[J].中国生物防治学报,2017,33(5):623-630.
[21]刘杰,姜玉英,曾娟.2013年我国黏虫发生特点分析[J].植物保护,2015,41(3):131-137.
[22]Zhang Y H,Zhang Z,Li C,et al.Seasonal migratory behavior of Mythimna separata(Lepidoptera:Noctuidae)in Northeast China[J].Acta Entomologica Sinica,2013,56(12):1418-1429.
[23]王玲,江幸福,罗礼智,等.粘虫类钙粘蛋白基因的克隆、序列分析及时空表达[J].昆虫学报,2011,54(10):1094-1103.
[24]Neumann C A,Krause D S,Carman C V,et al.Essential role for the peroxiredoxin Prdx1 in erythrocyte antioxidant defence and tumour suppression[J].Nature,2003,424(6948):561-565.
[25]Zhao X,Smartt C T,Li J,et al.Aedes aegypti peroxidase gene characterization and developmental expression[J].Insect Biochemistry and Molecular Biology,2001,31(4/5):481-490.
[26]Lushchak V I.Environmentally induced oxidative stress in aquatic animals[J].Aquatic Toxicology,2011,101(1):13-30.
[27]Kottuparambil S,Shin W,Brown M T,et al.UV-B affects photosynthesis,ROS production and motility of the freshwater flagellate,Euglena agilis Carter[J].Aquatic Toxicology,2012,s(122-123):206-213.
[28]Corona M,Robinson G E.Genes of the antioxidant system of the honey bee:Annotation and Phylogeny[J].Insect Molecular Biology,2006,15(5):687-701.
[29]Kobayashi-Miura M,Shioji K,Hoshino Y,et al.Oxygen sensing and redox signaling:the role of thioredoxin in embryonic development and cardiac diseases[J].American Journal of Physiology Heart&Circulatory Physiology,2007,292(5):2040-2050.
[30]李毅平,龚和.昆虫体内抗氧化系统研究进展[J].生命科学,1998(5):240-243.
[31]杨唐斌,梅尚筠.热休克对家蚕幼虫抗氧化酶活性的影响[J].生物化学与生物物理进展,1996(2):153-156.
[32]李庆,吴蕾,杨刚,等.温度和紫外辐射胁迫对西藏飞蝗抗氧化系统的影响[J].生态学报,2012,32(10):3189-3197.
[33]王艳敏,仵均祥,万方浩.昆虫对极端高低温胁迫的响应研究[J].环境昆虫学报,2010,32(2):250-255.
[34]庹瑞锐,赵运林,董萌,等.土壤重金属镉、铅污染的植物修复技术研究进展[J].贵州农业科学,2011,39(6):216-220.