低温胁迫对越冬期中华蜜蜂体内抗氧化指标及耐寒基因表达的影响
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摘要
低温环境造成蜜蜂的冷伤害,是养蜂生产中制约蜂群发展的主要因素之一。本试验旨在通过研究低温胁迫下越冬期中华蜜蜂(Apis cerana cerana)体内抗氧化指标及耐寒基因表达的变化,探索低温胁迫对蜜蜂的冷伤害机制。以中国北方越冬期的中华蜜蜂作为研究对象,选取蜂群中的越冬工蜂900只,随机分为6个组,1个对照组和5个试验组,每组3个重复,每个重复50只。5个试验组于实验室内饲养,于低温条件(16℃)下分别暴露15、30、45、60和90 min,对照组不在低温条件下暴露。暴露结束后各组取20只蜜蜂样品,立即置于液氮保存,用于测定蜂体总抗氧化能力(T-AOC)及超氧化物歧化酶(SOD)、过氧化物酶(POD)活性,并采用实时荧光定量PCR方法检测蜂体耐寒基因类致死必需蛋白[L(2) efl]、SOD、海藻糖-6-磷酸合成酶(TPS)、卵黄蛋白原(Vg)、过氧化氢酶(CAT)的相对表达量。结果显示:1)低温胁迫下,中华蜜蜂体内T-AOC及SOD、POD活性随着暴露时间的延长基本呈逐渐升高趋势,并均在低温暴露90 min时达到峰值,分别为(57.62±5.95) U/mL、(21.39±1.77) U/mL和(27.24±2.41) U/mL,与对照组差异显著(P<0.05)。2)低温胁迫下,随着暴露时间的延长,中华蜜蜂体内L(2) efl基因的相对表达量升高,TPS和SOD基因的相对表达量先升高后降低,而CAT基因的相对表达量则一直处于较低水平。与对照组相比,各试验组L(2) efl基因的相对表达量显著升高(P<0.05);低温暴露15和30 min组TPS基因的相对表达量显著升高(P<0.05);低温暴露30和45 min组Vg基因的相对表达量显著降低(P <0.05),而低温暴露60 min组Vg基因的相对表达量则显著升高(P<0.05);低温暴露15、30、45和60 min组CAT基因的相对表达量显著降低(P<0.05);低温暴露30、45 min组SOD基因的相对表达量显著升高(P<0.05)。由此得出,低温胁迫下越冬期中华蜜蜂抗氧化系统的防御能力趋强,耐寒基因L(2) efl、TPS和SOD表达上调,CAT表达下调,提示中华蜜蜂对低温胁迫存在耐寒响应的生理机制。
The cold damage caused by the lowtemperature environment is one of the main factors restricting the development of Apis cerana cerana colonies in beekeeping production. In this study,the changes of antioxidant indexes and cold-resistance gene expression of Apis cerana cerana during over-wintering period under lowtemperature stress were observed,and the cold damage mechanism of lowtemperature stress on bees was explored. Apis cerana cerana during the wintering period in northern China as the study subject. A total of 900 wintering worker bees in the bee colony were randomly divided into six groups including one control group and five test groups,each group had three replicates,and each replicate had fifty bees. Bees in the five test groups were kept in the laboratory,and exposed under lowtemperature (16 ℃) for 15,30,45,60 and 90 min,respectively,while bees in the control group without any treatment. After exposure,twenty bee samples were taken from each group and immediately placed in liquid nitrogen for storage. The total antioxidant capacity (T-AOC) and the activities of superoxide dismutase (SOD) and catalase (CAT) of bees were determined,and five cold-resistance genes protein lethal (2) essential for life [l (2) efl],SOD,trehalose-6-phosphate synthase (TPS),vitellogenin (Vg) and catalase (CAT) relative expression levels were detected by real-time fluorescent quantitative PCR method. The results showed as follows: 1) under the lowtemperature stress,the T-AOC and the activities of SOD and CAT of Apis cerana cerana gradually increased with the prolongation of lowtemperature exposure time (P<0.05),and all of them reached the highest after lowtemperature exposure 90 min,they were (57.62±5.95) U/mL, (21.39±1.77) U/mL and (27.24 ± 2.41) U/mL,respectively,and there were significant differences with the control group (P<0.05). 2) Under the lowtemperature stress,with the prolongation of lowtemperature exposure time,the relative expression level of L (2) efl gene of Apis cerana cerana was up-regulated,the relative expression levels of TPS and SOD genes were up-regulated first and then downregulated,while the relative expression level of CAT gene was in lower level constantly. Compared with the control group,the relative expression level of L (2) efl gene in five test groups was significantly increased (P<0.05),the relative expression level of TPS gene in lowtemperature exposure 15 and 30 min groups was significantly increased (P < 0. 05),the relative expression level of Vg gene in lowtemperature exposure 30 and45 min groups was significantly increased (P<0.05) and in lowtemperature exposure 60 min group was significantly decreased (P<0.05),the relative expression level of CAT gene in lowtemperature exposure 15,30,45 and 60 min groups was significantly decreased (P<0.05),and the relative expression level of SOD gene in lowtemperature exposure 15,30,45 and 60 min groups was significantly increased (P<0.05). In conclusion,under the lowtemperature stress,the defense ability of antioxidant system of Apis cerana cerana during overwintering period is stronger,cold-resistance genes L (2) efl,TPS and SOD are expressed in a higher level and CAT gene is expressed in a lower level. It is indicated that Apis cerana cerana to lowtemperature stress exists the physiological mechanism of cold tolerance response.
The cold damage caused by the lowtemperature environment is one of the main factors restricting the development of Apis cerana cerana colonies in beekeeping production. In this study,the changes of antioxidant indexes and cold-resistance gene expression of Apis cerana cerana during over-wintering period under lowtemperature stress were observed,and the cold damage mechanism of lowtemperature stress on bees was explored. Apis cerana cerana during the wintering period in northern China as the study subject. A total of 900 wintering worker bees in the bee colony were randomly divided into six groups including one control group and five test groups,each group had three replicates,and each replicate had fifty bees. Bees in the five test groups were kept in the laboratory,and exposed under lowtemperature (16 ℃) for 15,30,45,60 and 90 min,respectively,while bees in the control group without any treatment. After exposure,twenty bee samples were taken from each group and immediately placed in liquid nitrogen for storage. The total antioxidant capacity (T-AOC) and the activities of superoxide dismutase (SOD) and catalase (CAT) of bees were determined,and five cold-resistance genes protein lethal (2) essential for life [l (2) efl],SOD,trehalose-6-phosphate synthase (TPS),vitellogenin (Vg) and catalase (CAT) relative expression levels were detected by real-time fluorescent quantitative PCR method. The results showed as follows: 1) under the lowtemperature stress,the T-AOC and the activities of SOD and CAT of Apis cerana cerana gradually increased with the prolongation of lowtemperature exposure time (P<0.05),and all of them reached the highest after lowtemperature exposure 90 min,they were (57.62±5.95) U/mL, (21.39±1.77) U/mL and (27.24 ± 2.41) U/mL,respectively,and there were significant differences with the control group (P<0.05). 2) Under the lowtemperature stress,with the prolongation of lowtemperature exposure time,the relative expression level of L (2) efl gene of Apis cerana cerana was up-regulated,the relative expression levels of TPS and SOD genes were up-regulated first and then downregulated,while the relative expression level of CAT gene was in lower level constantly. Compared with the control group,the relative expression level of L (2) efl gene in five test groups was significantly increased (P<0.05),the relative expression level of TPS gene in lowtemperature exposure 15 and 30 min groups was significantly increased (P < 0. 05),the relative expression level of Vg gene in lowtemperature exposure 30 and45 min groups was significantly increased (P<0.05) and in lowtemperature exposure 60 min group was significantly decreased (P<0.05),the relative expression level of CAT gene in lowtemperature exposure 15,30,45 and 60 min groups was significantly decreased (P<0.05),and the relative expression level of SOD gene in lowtemperature exposure 15,30,45 and 60 min groups was significantly increased (P<0.05). In conclusion,under the lowtemperature stress,the defense ability of antioxidant system of Apis cerana cerana during overwintering period is stronger,cold-resistance genes L (2) efl,TPS and SOD are expressed in a higher level and CAT gene is expressed in a lower level. It is indicated that Apis cerana cerana to lowtemperature stress exists the physiological mechanism of cold tolerance response.
引文
[1]孟飞,胥保华,郭兴启.中华蜜蜂重要生物学特性相关功能基因研究进展[J].应用昆虫学报,2012,49(5):1338-1344.
[2]康乐.环境胁迫下的昆虫-植物相互关系[J].生态学杂志,1995,14(5):51-57,50.
[3]SINCLAIR B J,VERNON P,KLOK C J,et al.Insects at low temperatures:an ecological perspective[J].Trends in Ecology&Evolution,2003,18(5):257-262.
[4]ROJAS R R,LEOPOLD R A.Chilling injury in the housefly:evidence for the role of oxidative stress betw een pupariation and emergence[J].Cryobiology,1996,33(4):447-458.
[5]常志光,牛庆生,闫德斌,等.不同蜜蜂品种越冬后期血淋巴总SOD活力与抗寒力的相关性研究[J].中国蜂业,2015,66(3):16-18.
[6]赵静,李姝,陈珍珍,王甦,等.低温胁迫对异色瓢虫成虫存活及体内几种酶活力的影响[J].植物保护学报,2014,41(5):513-518.
[7]GHISELLI A,SERAFINI M,NATELLA F,et al.Total antioxidant capacity as a tool to assess redox status:critical view and experimental data[J].Free Radical Biology and M edicine,2000,29(11):1106-1114.
[8]张青,涂永勤,刘怀,等.温度胁迫对小金蝠蛾幼虫抗氧化酶活性的影响[J].环境昆虫学报,2016,38(1):47-53.
[9]HOFFMANN A A,SRENSEN J G,LOESCHCKEV.Adaptation of Drosophila to temperature extremes:bringing together quantitative and molecular approaches[J].Journal of Thermal Biology,2003,28(3):175-216.
[10]HUANG L H,WANG C Z,KANG L.Cloning and expression of five heat shock protein genes in relation to cold hardening and development in the leafminer,Liriomyza sativa[J].Journal of Insect Physiology,2009,55(3):279-285.
[11]KURZIK-DUMKE U,LOHMANN E.Sequence of the new Drosophila melanogaster small heat-shock-related gene,lethal(2)essential for life[L(2)efl],at locus59F4,5[J].Gene,1995,154(2):171-175.
[12]秦资.异色瓢虫TPS与Treh1基因低温诱导表达分析[D].硕士学位论文.杭州:杭州师范大学,2012.
[13]AMDAM G V,NORBERG K,OMHOLT S W,et al.Higher vitellogenin concentrations in honey bee w orkers may be an adaptation to life in temperate climates[J].Insectes Sociaux,2005,52(4):316-319.
[14]赵晓峰,刘新生,李化龙,等.冬季外界环境温度变化对中、意蜜蜂箱内温度影响的试验分析[J].陕西农业科学,2008,54(4):35-38.
[15]LEE R E,Jr,CHEN C P,DENLINGER D L.A rapid cold-hardening process in insects[J].Science,1987,238(4832):1415-1417.
[16]ZACHARIASSEN K E,KRISTIANSEN E,PEDERS-EN S A.Inorganic ions in cold-hardiness[J].Cryobiology,2004,48(2):126-133.
[17]OVERGAARD J,SRENSEN J G,PETERSEN S O,et al.Changes in membrane lipid composition following rapid cold hardening in Drosophila melanogaster[J].Journal of Insect Physiology,2005,51(11):1173-1182.
[18]李周直,沈惠娟,蒋巧很,等.几种昆虫体内保护酶系统活力的研究[J].昆虫学报,1994,37(4):399-403.
[19]刘井兰,于建飞,吴进才,等.昆虫活性氧代谢[J].应用昆虫学报,2006,43(6):752-756.
[20]于平.超氧化物歧化酶研究进展[J].生物学通报,2006,41(1):4-6.
[21]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):956-964.
[22]郑玉涛.高温胁迫对西花蓟马抗氧化酶活性的影响及CAT基因的克隆与表达[D].硕士学位论文.扬州:扬州大学,2015.
[23]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.
[24]SHIGEOKA S,ISHIKAWA T,TAMOI M,et al.Regulation and function of ascorbate peroxidase isoenzymes[J].Journal of Experimental Botany,2002,53(372):1305-1319.
[25]KHACHIK F,CARVALHO L,BERNSTEIN P S,et al.Chemistry,distribution,and metabolism of tomato carotenoids and their impact on human health[J].Experimental Biology and M edicine,2002,227(10):845-851.
[26]YILDIRIM,BYKBINGL Z.Effects of supplementation w ith a combination of cobalt and ascorbic acid on antioxidant enzymes and lipid peroxidation levels in streptozocin-diabetic rat liver[J].Biological Trace Element Research,2002,90(1/2/3):143-154.
[27]张卫星,胥保华.蜜蜂卵黄原蛋白的研究进展[J].蜜蜂杂志,2014,34(5):5-7.
[28]ZHANG W X,LIU Z G,ZHU M,et al.Molecular cloning,expression and oxidative stress response of the vitellogenin G gene(AccVg)from Apis cerana cerana[J].Apidologie,2017,48(5):599-611.
[29]REINEKE A.Identification and expression of a small heat shock protein in tw o lines of the endoparasitic w asp Venturia canescens[J].Comparative Biochemistry and Physiology Part A:M olecular&Integrative Physiology,2005,141(1):60-69.
[30]ZHANG Y Y,LIU Y L,GUO X L,et al.SHsp22.6,an intronless small heat shock protein gene,is involved in stress defence and development in Apis cerana cerana[J].Insect Biochemistry and Molecular Biology,2014,53:1-12.
[31]LACHTA M,BERKOVP,VAMBERA J,et al.Physiology of cold-acclimation in non-diapausing adults of Pyrrhocoris apterus(Heteroptera)[J].European Journal of Entomology,2013,99(2):181-187.
[32]于彩虹,卢丹,林荣华,等.海藻糖---昆虫的血糖[J].应用昆虫学报,2008,45(5):832-837.
[33]秦加敏,罗术东,和绍禹,等.昆虫海藻糖与海藻糖酶的特性及功能研究[J].环境昆虫学报,2015,37(1):163-169.
[34]史彩华,胡静荣,李传仁,等.环境胁迫下昆虫的耐寒适应机制研究进展[J].植物保护,2016,42(6):21-28.
[35]KLOWDEN M J.Physiological systems in insects[M].3rd ed.Amsterdam:Academic Press,2013.
[36]秦资.异色瓢虫TPS与Treh1基因低温诱导表达分析[D].硕士学位论文.杭州:杭州师范大学,2012.
[37]王建华,刘鸿先,徐同.超氧物歧化酶(SOD)在植物逆境和衰老生理中的作用[J].植物生理学通讯,1989(1):1-7.
[38]AHMAD S,DUVAL D L,WEINHOLD L C,et al.Cabbage looper antioxidant enzymes:tissue specificity[J].Insect Biochemistry,1991,21(5):563-572.
[39]吴启仙,夏嫱,重金属对昆虫抗氧化酶影响研究进展[J].环境昆虫学报,2014,36(2):247-251.
[2]康乐.环境胁迫下的昆虫-植物相互关系[J].生态学杂志,1995,14(5):51-57,50.
[3]SINCLAIR B J,VERNON P,KLOK C J,et al.Insects at low temperatures:an ecological perspective[J].Trends in Ecology&Evolution,2003,18(5):257-262.
[4]ROJAS R R,LEOPOLD R A.Chilling injury in the housefly:evidence for the role of oxidative stress betw een pupariation and emergence[J].Cryobiology,1996,33(4):447-458.
[5]常志光,牛庆生,闫德斌,等.不同蜜蜂品种越冬后期血淋巴总SOD活力与抗寒力的相关性研究[J].中国蜂业,2015,66(3):16-18.
[6]赵静,李姝,陈珍珍,王甦,等.低温胁迫对异色瓢虫成虫存活及体内几种酶活力的影响[J].植物保护学报,2014,41(5):513-518.
[7]GHISELLI A,SERAFINI M,NATELLA F,et al.Total antioxidant capacity as a tool to assess redox status:critical view and experimental data[J].Free Radical Biology and M edicine,2000,29(11):1106-1114.
[8]张青,涂永勤,刘怀,等.温度胁迫对小金蝠蛾幼虫抗氧化酶活性的影响[J].环境昆虫学报,2016,38(1):47-53.
[9]HOFFMANN A A,SRENSEN J G,LOESCHCKEV.Adaptation of Drosophila to temperature extremes:bringing together quantitative and molecular approaches[J].Journal of Thermal Biology,2003,28(3):175-216.
[10]HUANG L H,WANG C Z,KANG L.Cloning and expression of five heat shock protein genes in relation to cold hardening and development in the leafminer,Liriomyza sativa[J].Journal of Insect Physiology,2009,55(3):279-285.
[11]KURZIK-DUMKE U,LOHMANN E.Sequence of the new Drosophila melanogaster small heat-shock-related gene,lethal(2)essential for life[L(2)efl],at locus59F4,5[J].Gene,1995,154(2):171-175.
[12]秦资.异色瓢虫TPS与Treh1基因低温诱导表达分析[D].硕士学位论文.杭州:杭州师范大学,2012.
[13]AMDAM G V,NORBERG K,OMHOLT S W,et al.Higher vitellogenin concentrations in honey bee w orkers may be an adaptation to life in temperate climates[J].Insectes Sociaux,2005,52(4):316-319.
[14]赵晓峰,刘新生,李化龙,等.冬季外界环境温度变化对中、意蜜蜂箱内温度影响的试验分析[J].陕西农业科学,2008,54(4):35-38.
[15]LEE R E,Jr,CHEN C P,DENLINGER D L.A rapid cold-hardening process in insects[J].Science,1987,238(4832):1415-1417.
[16]ZACHARIASSEN K E,KRISTIANSEN E,PEDERS-EN S A.Inorganic ions in cold-hardiness[J].Cryobiology,2004,48(2):126-133.
[17]OVERGAARD J,SRENSEN J G,PETERSEN S O,et al.Changes in membrane lipid composition following rapid cold hardening in Drosophila melanogaster[J].Journal of Insect Physiology,2005,51(11):1173-1182.
[18]李周直,沈惠娟,蒋巧很,等.几种昆虫体内保护酶系统活力的研究[J].昆虫学报,1994,37(4):399-403.
[19]刘井兰,于建飞,吴进才,等.昆虫活性氧代谢[J].应用昆虫学报,2006,43(6):752-756.
[20]于平.超氧化物歧化酶研究进展[J].生物学通报,2006,41(1):4-6.
[21]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):956-964.
[22]郑玉涛.高温胁迫对西花蓟马抗氧化酶活性的影响及CAT基因的克隆与表达[D].硕士学位论文.扬州:扬州大学,2015.
[23]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.
[24]SHIGEOKA S,ISHIKAWA T,TAMOI M,et al.Regulation and function of ascorbate peroxidase isoenzymes[J].Journal of Experimental Botany,2002,53(372):1305-1319.
[25]KHACHIK F,CARVALHO L,BERNSTEIN P S,et al.Chemistry,distribution,and metabolism of tomato carotenoids and their impact on human health[J].Experimental Biology and M edicine,2002,227(10):845-851.
[26]YILDIRIM,BYKBINGL Z.Effects of supplementation w ith a combination of cobalt and ascorbic acid on antioxidant enzymes and lipid peroxidation levels in streptozocin-diabetic rat liver[J].Biological Trace Element Research,2002,90(1/2/3):143-154.
[27]张卫星,胥保华.蜜蜂卵黄原蛋白的研究进展[J].蜜蜂杂志,2014,34(5):5-7.
[28]ZHANG W X,LIU Z G,ZHU M,et al.Molecular cloning,expression and oxidative stress response of the vitellogenin G gene(AccVg)from Apis cerana cerana[J].Apidologie,2017,48(5):599-611.
[29]REINEKE A.Identification and expression of a small heat shock protein in tw o lines of the endoparasitic w asp Venturia canescens[J].Comparative Biochemistry and Physiology Part A:M olecular&Integrative Physiology,2005,141(1):60-69.
[30]ZHANG Y Y,LIU Y L,GUO X L,et al.SHsp22.6,an intronless small heat shock protein gene,is involved in stress defence and development in Apis cerana cerana[J].Insect Biochemistry and Molecular Biology,2014,53:1-12.
[31]LACHTA M,BERKOVP,VAMBERA J,et al.Physiology of cold-acclimation in non-diapausing adults of Pyrrhocoris apterus(Heteroptera)[J].European Journal of Entomology,2013,99(2):181-187.
[32]于彩虹,卢丹,林荣华,等.海藻糖---昆虫的血糖[J].应用昆虫学报,2008,45(5):832-837.
[33]秦加敏,罗术东,和绍禹,等.昆虫海藻糖与海藻糖酶的特性及功能研究[J].环境昆虫学报,2015,37(1):163-169.
[34]史彩华,胡静荣,李传仁,等.环境胁迫下昆虫的耐寒适应机制研究进展[J].植物保护,2016,42(6):21-28.
[35]KLOWDEN M J.Physiological systems in insects[M].3rd ed.Amsterdam:Academic Press,2013.
[36]秦资.异色瓢虫TPS与Treh1基因低温诱导表达分析[D].硕士学位论文.杭州:杭州师范大学,2012.
[37]王建华,刘鸿先,徐同.超氧物歧化酶(SOD)在植物逆境和衰老生理中的作用[J].植物生理学通讯,1989(1):1-7.
[38]AHMAD S,DUVAL D L,WEINHOLD L C,et al.Cabbage looper antioxidant enzymes:tissue specificity[J].Insect Biochemistry,1991,21(5):563-572.
[39]吴启仙,夏嫱,重金属对昆虫抗氧化酶影响研究进展[J].环境昆虫学报,2014,36(2):247-251.