短时高温暴露下东亚飞蝗雌虫血淋巴蛋白表达分析及质谱鉴定
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摘要
为研究短时高温对东亚飞蝗Locusta migratoria manilensis Meyen血淋巴蛋白的影响,采用Bradford法、SDSPAGE电泳和质谱等方法,对东亚飞蝗雌虫血淋巴样品进行检测。结果表明:短时高温对血淋巴蛋白含量有显著影响(P<0.01),36℃-42℃范围内,随温度升高,血淋巴蛋白浓度亦升高,其中39℃、42℃处理组与对照组差异显著(P<0.01);短时高温对血淋巴蛋白种类存在一定影响,对照组雌虫血淋巴中存在11种蛋白,高温处理后,4种蛋白含量逐渐增加,6种蛋白含量没有明显变化,1种蛋白消失;经质谱检测,鉴定了5种蛋白,分别为载脂蛋白前体、酚氧化酶原、2个储存蛋白和19 kDa血淋巴蛋白,另外6条蛋白未被鉴定。推测载脂蛋白前体、酚氧化酶原、储存蛋白在东亚飞蝗应对高温胁迫过程中发挥重要作用。
In order to study the effect of short-term exposure to high temperature on proteins in hemolymph of Locusta migratoria manilensis Meyen,proteins in hemolymph of female adult locust were detected by Bradford assay,SDS-PAGE electrophoresis and MALDI-TOF-TOF/MS analysis.The results showed that short-term exposure to high temperature did a significant effect on concentration of protein in hemolymph(P<0.01).The concentration was increased with increasing temperature in the range of 36℃-42℃.There had significant increase on concentration between the treatment group(39℃,42℃) and the control(P<0.01).Short-term exposure to high temperature affected types of proteins in hemolymph.There had 11 types of proteins in hemolymph in the control.With increasing temperature,the concentration of 4 types of proteins were increased while 6 types of proteins showed no changed.Under different temperature treatments,1 type of proteins was disappeared.By MALDI-TOF-TOF/MS analysis,5 types of proteins were identified as apolipophorin precursor protein,pro-phenoloxidase 2,hexamerin-like protein 1,hexamerin-like protein 2 and basic 19 kDa hemolymph protein.The other 6 types of proteins were not identified.Apolipophorin precursor protein,pro-phenoloxidase,hexamerin protein may play pivotal roles in response to high temperature stress.
In order to study the effect of short-term exposure to high temperature on proteins in hemolymph of Locusta migratoria manilensis Meyen,proteins in hemolymph of female adult locust were detected by Bradford assay,SDS-PAGE electrophoresis and MALDI-TOF-TOF/MS analysis.The results showed that short-term exposure to high temperature did a significant effect on concentration of protein in hemolymph(P<0.01).The concentration was increased with increasing temperature in the range of 36℃-42℃.There had significant increase on concentration between the treatment group(39℃,42℃) and the control(P<0.01).Short-term exposure to high temperature affected types of proteins in hemolymph.There had 11 types of proteins in hemolymph in the control.With increasing temperature,the concentration of 4 types of proteins were increased while 6 types of proteins showed no changed.Under different temperature treatments,1 type of proteins was disappeared.By MALDI-TOF-TOF/MS analysis,5 types of proteins were identified as apolipophorin precursor protein,pro-phenoloxidase 2,hexamerin-like protein 1,hexamerin-like protein 2 and basic 19 kDa hemolymph protein.The other 6 types of proteins were not identified.Apolipophorin precursor protein,pro-phenoloxidase,hexamerin protein may play pivotal roles in response to high temperature stress.
引文
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Daniel AH,Diana EW.Presence of a single abundant storage hexamerin in both larvae and adults of the grasshopper,Schistocerca americana[J].Journal of Insect Physiology,2003,49(12):1189-1197.
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Guan J.Analysis of Proteomic of the Hemolyph in Moth in Silkworm,Bombyx mori[D].Chongqing:Southwest University,2007.[官建.家蚕羽化变态时期血液蛋白质组学研究[D].重庆:西南大学,2007]
Jiang FZ,Zheng LY,Guo JX,et al.Effects of temperature stress on insect fertility and its physiological and biochemical mechanisms[J].Journal of Environmental Entomology,2015,37(3):653-663.[蒋丰泽,郑灵燕,郭技星,等.温度对昆虫繁殖力的影响及其生理生化机制[J].环境昆虫学报,2015,37(3):653-663]
Kawooya JK,Osir EO,Law JH.Uptake of the major hemolymph lipoprotein and its transformation in the insect egg[J].Journal of Biological Chemistry,1988,263(18):8740-8747.
Lage C,Luel LB,Kenneth S.The pro PO-system:Pros and cons for its role in invertebrate immunity[J].Immunology,2008,29(6):263-271.
Li LS.The Influence on the Immunocompetence and Physiological Property of Locusta migratoria manilensis(Meyen)Parasitized by Blaesoxipha lapidosa[D].Hangzhou:Hangzhou Normal University,2012.[李灵顺.线纹折麻蝇寄生对东亚飞蝗免疫及生理特性的影响研究[D].杭州:杭州师范大学,2012]
Li S,Cai MT,Ma WY,et al.Difference in heat tolerance and enzyme activity between Calliptamus italicus(Orthopera:Acrididae)and Gomphocerus sibiricus(Orthopera:Acrididae)[J].Chinese Journal of Applied Entomology,2016,53(5):1077-1083.[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]
Ma G,Ma CS.The impacts of extreme high temperature on insect populations under climate change:A review[J].Science China Press:Scientia Sinica Vitae,2016,46(5):556-564.[马罡,马春森.气候变化下极端高温对昆虫种群影响的研究进展[J].中国科学:生命科学,2016,46(5):556-564]
Pan F,Chen MC,Xiao TB,et al.Research advances on effect of variable temperature on growth,development and reproduction of insect[J].Journal of Environmental Entomology,2014,36(2):240-246.[潘飞,陈绵才,肖彤斌,等.变温对昆虫生长发育和繁殖影响的研究进展[J].环境昆虫学报,2014,36(2):240-246]
Sevala VL,Bagnères AG,Kuenzli M,et al.Cuticular hydrocarbons of the dampwood termite,Zootermopsis nevadensis:Caste differences and role of lipophorin in transport of hydrocarbons and hydrocarbon metabolites[J].Journal of Chemical Ecology,2000,26(3):765-789.
StefanR,Dim C.Increase of extreme events in a warming world[J].Proceedings of the National Academy of Sciences,2011,108(44):17905-17909.
Tang B,Wang SG,Zhang F.Two storage hexamerins from the beet armyworm Spodoptera exigua:Cloning,characterization and the effect of gene silencing on survival[J].BMC Molecular Biology,2010,11(1):1-13.
Tao SX,Tian YZ,Xiao Y,et al.Effects of temperature on development and humoral immune defense of Tenebrio molitor(Coleoptera:Tenebrionidae)larvae[J].Acta Entomologica Sinica,2014,57(5):616-620.[陶淑霞,田宇璋,肖月,等.温度对黄粉虫幼虫生长发育及体液免疫防御能力的影响[J].昆虫学报,2014,57(5):616-620]
Tu XB,Gao S,Yasen·Shali,et al.Effects on eating and food utilization efficiency of Locusta migratoria manilensis at different temperatures[J].Chinese Bulletin of Entomology,2010,47(4):690-693.[涂雄兵,高松,牙森·沙力,等.温度对东亚飞蝗取食影响及其食物利用效率研究[J].昆虫知识,2010,47(4):690-693]
Tu XB,Wang GJ,Nong XQ,et al.Mating and ovipositing behaviors of Locusta migratoria manilensis at different temperatures[J].Plant Protection,2011,37(4):124-125,137.[涂雄兵,王广君,农向群,等.不同温度下东亚飞蝗交配及产卵行为[J].植物保护,2011,37(4):124-125,137]
Wang CT.Analysis of the Differentially-expressed Glycoproteins in Locusta migratoria Haemolymph Mycosed with Metarhizium anisopliae and the Phosphoproteins in the Haemolymph Targetted by the Fungal Protein Tyrosine Phosphatase[D].Chongqing:Chongqing University,2007.[王楚桃.绿僵菌侵染蝗虫差异糖蛋白及其酪氨酸蛋白磷酸酶在寄主血淋巴中的靶标蛋白分析[D].重庆:重庆大学,2007]
Xiang M,Fan TS,Hu HX,et al.Effects of short-term exposure to high temperature on the survival and fecundity of Calliptamus italicus(Orthopera:Acrididae)[J].Chinese Journal of Applied Entomology,2017,54(3):426-433.[向敏,樊泰山,扈鸿霞,等.短时高温对意大利蝗存活和生殖的影响[J].应用昆虫学报,2017,54(3):426-433]
Xu ZS.Effects of Temperature on Storage Proteins of Bradysia odoriphaga and of Soil Water Content on the Survival and Reproduction of the Pest[D].Taian:Shandong Agricultural University,2015.[徐志松.温度对韭菜迟眼蕈蚊储存蛋白的影响和土壤含水量对该害虫生存与生殖的影响[D].泰安:山东农业大学,2015.
Yue M,Lei ZR,Zhu BZ,et al.High temperature tolerance and thermoregulation behavior of the oriental migratory locust,Locusta migratoria manilensis(Orthoptera:Acrididae)[J].Acta Entomologica Sinica,2009,52(10):1103-1109.[岳梅,雷仲仁,朱彬洲,等.东亚飞蝗耐高温能力及其体温调节行为[J].昆虫学报,2009,52(10):1103-1109]
Zhang XH,Li YL,Zhang KJ,et al.Cloning and expression analysis of the hexamerin subunit type 2(Hex2)gene from the grasshopper Calliptamus italicus(Orthoptera:Catantopidae)[J].Acta Entomologica Sinica,2016,59(2):156-163.[张晓红,李艳丽,张凯建,等.意大利蝗存储蛋白Hex2基因的克隆与表达分析(英文)[J].昆虫学报,2016,59(2):156-163]
Zhao Z,Li XK,Jin LB,et al.Temperature effect on phenoloxidase activity in different stage of Locusta migratoria manilensis Meyen[J]Journal of Jilin Agricultural University,2011,33(6):613-616.[赵卓,李校堃,金立波,等.温度对不同发育阶段东亚飞蝗酚氧化酶活性的影响[J].吉林农业大学学报,2011,33(6):613-616]
Bradford MM.A rapid and sensitive method for the quantitation o microgram quantities of protein utilizing the principle of proteindye binding[J].Analytical Biochemistry,1976,72(1/2):248-254.
Chen TT,Strahlendorf PW,Wyatt GR.Vitellin and vitellogenin from locusts(Locusta migratoria),properties and post-translational modification in the fat body[J].The Journal of Biological Chemistry,1978,253(5):5325-5331.
Chen WZ,Li B,Gong CL,et al.The research on the activity of phenoloxidase in the haemolymph of Bombyx mori and its influence factors[J].Jiangsu Sericulture,2005,2:12-15.[陈文柱,李兵,贡成良,等.酚氧化酶在家蚕血淋巴中的活力分布及影响因素的研究[J].江苏蚕业,2005,2:12-15]
Daniel AH,Diana EW.Presence of a single abundant storage hexamerin in both larvae and adults of the grasshopper,Schistocerca americana[J].Journal of Insect Physiology,2003,49(12):1189-1197.
Dong LJ,Zhang XH,Li YL,et al.Cloning and expression analysis of a hexamerin gene from Acrida cinerea(Acridoidea:Acrididae)[J].Journal of Agricultural Science and Technology,2015,17(4):78-84.[董丽君,张晓红,李艳丽,等.中华剑角蝗储存蛋白基因的克隆及表达分析[J].中国农业科技导报,2015,17(4):78-84]
Du Y,Ma CS,Zhao QH,et al.Effects of heat stress on physiological and biochemical mechanisms of insects:A literature review[J].Acta Ecologica Sinica,2007,27(4):1565-1572.[杜尧,马春森,赵清华,等.高温对昆虫影响的生理生化作用机理研究进展[J].生态学报,2007,27(4):1565-1572]
Gao SJ,Han HB,Wang N,et al.Influences of temperature and humidity conditions on flight capacity and main utilization of energy substances in Oedaleusasi aticus[J].Pratacultural Science,2016,33(7):1410-1417.[高书晶,韩海斌,王宁,等.温湿度对亚洲小车蝗飞行能力及主要能源物质利用的影响[J].草业科学,2016,33(7):1410-1417]
Guan J.Analysis of Proteomic of the Hemolyph in Moth in Silkworm,Bombyx mori[D].Chongqing:Southwest University,2007.[官建.家蚕羽化变态时期血液蛋白质组学研究[D].重庆:西南大学,2007]
Jiang FZ,Zheng LY,Guo JX,et al.Effects of temperature stress on insect fertility and its physiological and biochemical mechanisms[J].Journal of Environmental Entomology,2015,37(3):653-663.[蒋丰泽,郑灵燕,郭技星,等.温度对昆虫繁殖力的影响及其生理生化机制[J].环境昆虫学报,2015,37(3):653-663]
Kawooya JK,Osir EO,Law JH.Uptake of the major hemolymph lipoprotein and its transformation in the insect egg[J].Journal of Biological Chemistry,1988,263(18):8740-8747.
Lage C,Luel LB,Kenneth S.The pro PO-system:Pros and cons for its role in invertebrate immunity[J].Immunology,2008,29(6):263-271.
Li LS.The Influence on the Immunocompetence and Physiological Property of Locusta migratoria manilensis(Meyen)Parasitized by Blaesoxipha lapidosa[D].Hangzhou:Hangzhou Normal University,2012.[李灵顺.线纹折麻蝇寄生对东亚飞蝗免疫及生理特性的影响研究[D].杭州:杭州师范大学,2012]
Li S,Cai MT,Ma WY,et al.Difference in heat tolerance and enzyme activity between Calliptamus italicus(Orthopera:Acrididae)and Gomphocerus sibiricus(Orthopera:Acrididae)[J].Chinese Journal of Applied Entomology,2016,53(5):1077-1083.[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]
Ma G,Ma CS.The impacts of extreme high temperature on insect populations under climate change:A review[J].Science China Press:Scientia Sinica Vitae,2016,46(5):556-564.[马罡,马春森.气候变化下极端高温对昆虫种群影响的研究进展[J].中国科学:生命科学,2016,46(5):556-564]
Pan F,Chen MC,Xiao TB,et al.Research advances on effect of variable temperature on growth,development and reproduction of insect[J].Journal of Environmental Entomology,2014,36(2):240-246.[潘飞,陈绵才,肖彤斌,等.变温对昆虫生长发育和繁殖影响的研究进展[J].环境昆虫学报,2014,36(2):240-246]
Sevala VL,Bagnères AG,Kuenzli M,et al.Cuticular hydrocarbons of the dampwood termite,Zootermopsis nevadensis:Caste differences and role of lipophorin in transport of hydrocarbons and hydrocarbon metabolites[J].Journal of Chemical Ecology,2000,26(3):765-789.
StefanR,Dim C.Increase of extreme events in a warming world[J].Proceedings of the National Academy of Sciences,2011,108(44):17905-17909.
Tang B,Wang SG,Zhang F.Two storage hexamerins from the beet armyworm Spodoptera exigua:Cloning,characterization and the effect of gene silencing on survival[J].BMC Molecular Biology,2010,11(1):1-13.
Tao SX,Tian YZ,Xiao Y,et al.Effects of temperature on development and humoral immune defense of Tenebrio molitor(Coleoptera:Tenebrionidae)larvae[J].Acta Entomologica Sinica,2014,57(5):616-620.[陶淑霞,田宇璋,肖月,等.温度对黄粉虫幼虫生长发育及体液免疫防御能力的影响[J].昆虫学报,2014,57(5):616-620]
Tu XB,Gao S,Yasen·Shali,et al.Effects on eating and food utilization efficiency of Locusta migratoria manilensis at different temperatures[J].Chinese Bulletin of Entomology,2010,47(4):690-693.[涂雄兵,高松,牙森·沙力,等.温度对东亚飞蝗取食影响及其食物利用效率研究[J].昆虫知识,2010,47(4):690-693]
Tu XB,Wang GJ,Nong XQ,et al.Mating and ovipositing behaviors of Locusta migratoria manilensis at different temperatures[J].Plant Protection,2011,37(4):124-125,137.[涂雄兵,王广君,农向群,等.不同温度下东亚飞蝗交配及产卵行为[J].植物保护,2011,37(4):124-125,137]
Wang CT.Analysis of the Differentially-expressed Glycoproteins in Locusta migratoria Haemolymph Mycosed with Metarhizium anisopliae and the Phosphoproteins in the Haemolymph Targetted by the Fungal Protein Tyrosine Phosphatase[D].Chongqing:Chongqing University,2007.[王楚桃.绿僵菌侵染蝗虫差异糖蛋白及其酪氨酸蛋白磷酸酶在寄主血淋巴中的靶标蛋白分析[D].重庆:重庆大学,2007]
Xiang M,Fan TS,Hu HX,et al.Effects of short-term exposure to high temperature on the survival and fecundity of Calliptamus italicus(Orthopera:Acrididae)[J].Chinese Journal of Applied Entomology,2017,54(3):426-433.[向敏,樊泰山,扈鸿霞,等.短时高温对意大利蝗存活和生殖的影响[J].应用昆虫学报,2017,54(3):426-433]
Xu ZS.Effects of Temperature on Storage Proteins of Bradysia odoriphaga and of Soil Water Content on the Survival and Reproduction of the Pest[D].Taian:Shandong Agricultural University,2015.[徐志松.温度对韭菜迟眼蕈蚊储存蛋白的影响和土壤含水量对该害虫生存与生殖的影响[D].泰安:山东农业大学,2015.
Yue M,Lei ZR,Zhu BZ,et al.High temperature tolerance and thermoregulation behavior of the oriental migratory locust,Locusta migratoria manilensis(Orthoptera:Acrididae)[J].Acta Entomologica Sinica,2009,52(10):1103-1109.[岳梅,雷仲仁,朱彬洲,等.东亚飞蝗耐高温能力及其体温调节行为[J].昆虫学报,2009,52(10):1103-1109]
Zhang XH,Li YL,Zhang KJ,et al.Cloning and expression analysis of the hexamerin subunit type 2(Hex2)gene from the grasshopper Calliptamus italicus(Orthoptera:Catantopidae)[J].Acta Entomologica Sinica,2016,59(2):156-163.[张晓红,李艳丽,张凯建,等.意大利蝗存储蛋白Hex2基因的克隆与表达分析(英文)[J].昆虫学报,2016,59(2):156-163]
Zhao Z,Li XK,Jin LB,et al.Temperature effect on phenoloxidase activity in different stage of Locusta migratoria manilensis Meyen[J]Journal of Jilin Agricultural University,2011,33(6):613-616.[赵卓,李校堃,金立波,等.温度对不同发育阶段东亚飞蝗酚氧化酶活性的影响[J].吉林农业大学学报,2011,33(6):613-616]