UV胁迫下棉铃虫生理生化响应及蛋白质组学研究
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摘要
棉铃虫Helicoverpa armigera (Hiiber)隶属于鳞翅目(Lepioloptera)夜蛾科(Noctuidae),是我国乃至世界上的重要农业害虫。棉铃虫是一种典型的趋光性昆虫,对紫外光(Ultraviolet, UV)十分敏感,本研究以棉铃虫为试验材料,研究UV照射下其体内生理生化响应及差异蛋白质组的变化。主要研究结果如下：
1.棉铃虫抗氧化系统对UV胁迫的响应
为了验证UV照射会对昆虫造成氧化胁迫这一假设,本研究检测了UV照射不同时间(0、30、60和90min)对棉铃虫总抗氧化能力的影响；通过测定过氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POX)、谷胱甘肽-S-转移酶(GST)及谷胱甘肽还原酶(GR)活性,分析了棉铃虫体内酶促抗氧化系统对UV胁迫的应答反应。当UV照射棉铃虫成虫30 min时,其体内总抗氧化能力、SOD、CAT、POX和GST活性均显著升高。当照射时间延长至60和90 min时,CAT和GST活性仍显著高于对照组水平,但总抗氧化能力和SOD活性恢复到对照组水平,POX活性则明显降低。本试验证实了UV照射能够对棉铃虫造成氧化胁迫的假设,提出UV照射对趋光性昆虫是一种光胁迫的理论。
2.UV胁迫对棉铃虫乙酰胆碱酯酶和羧酸酯酶活性的影响
本试验探讨了UV胁迫与棉铃虫体内乙酰胆碱酯酶(AChE)及羧酸酯酶(CarE)活性之间的关系,分析比较了两种酶活性在UV照射不同时间(0、30、60和90 min)下的变化情况。棉铃虫经过UV照射不同时间后,体内的AChE活性均有所降低,且在30 min时达到显著水平。UV照射对棉铃虫体内的CarE活性也产生了一定的抑制作用,与对照组相比,照射时间为30、60和90min的UV处理组CarE活性均显著低于对照水平。本研究推测,昆虫体内的AChE和CarE活性除了受已报道的杀虫剂的影响外,也有可能随着其它胁迫环境的作用而发生改变,如UV胁迫。
3.UV胁迫对棉铃虫几种同工酶的影响
为了分析UV胁迫条件下棉铃虫体内酯酶同工酶、POX同工酶和CAT同工酶的变化,本研究采用了同工酶电泳方法。与对照相比,UV处理组酯酶同工酶谱带发生了质和量的变化。照射时间为30和60 min时,谱带E4、E9、E10增强,谱带E2、E8减弱,谱带E1、E5、E7、E11消失,新增了E3、E6谱带；照射时间延长至90 min时,谱带E4、E9增强,谱带E2、E8减弱,谱带E1、E5、E7消失,新增了E3、E6谱带。棉铃虫POX同工酶主要有6条谱带,与对照组相比,棉铃虫P5谱带在UV照射处理下增强。棉铃虫CAT同工酶主要有2条谱带,在经过UV照射不同时间后谱带变化有所不同,与对照组相比,UV处理组的P1谱带增强,而P2谱带在30 min时减弱,60 min时恢复到对照水平,90 min时与对照相比又有所减弱。本研究推测这些同工酶谱带发生的变化,可能是昆虫对UV胁迫条件的一种适应反应。
4.棉铃虫响应UV胁迫的蛋白质组研究
为了研究UV胁迫条件下棉铃虫体内蛋白的变化,本文采用了比较蛋白质组学的方法。分别提取正常条件和UV照射1h条件下的棉铃虫体内总蛋白,然后进行双向电泳分离虫体的总蛋白,共检测到至少1200个蛋白点,其中21个被下调,12个被上调。质谱分析共鉴定出29个蛋白点,包括丝裂原活化蛋白激酶、Ste20类激酶CG40293-PA亚型A、ADP-核糖基化因子鸟苷酸转换因子2(BFA抑制)亚型CRA a、锌指蛋白768、预测的PPAR-α相互作用复合蛋白285相似蛋白、牦牛儿基转移酶Ⅰβ亚基、推测的线粒体辛酰基转移酶、热激蛋白21.4、预测的CAT相似蛋白、烯醇酶、H转运ATP合成酶β亚基亚型1、H转运ATP合成酶β亚基亚型2、异戊烯基半胱氨酸氧化酶1、腺苷脱氨酶相似蛋白A、3-羟-3-甲基戊二酰辅酶A还原酶、法呢基焦磷酸合成酶、肌动蛋白、肌球蛋白重链、肌肉肌球蛋白重链、嗅觉受体类似物、溶菌酶C、推测的染色体结构维持蛋白和一些功能未知的假设蛋白。这些蛋白多数具有重要的生物学功能,参与了信号转导、RNA加工、蛋白质加工、氧化还原平衡、代谢、及细胞骨架稳定等过程。
The cotton bollworm, Helicoverpa armigera (Hubner) (Lepidoptera:Noctuidae) is one of the most serious insect pest in China and neighboring contries. The moths of this nocturnal insect display a conspicuous positive phototactic behaviour to light stimuli, and are especially sensitive to Ultraviolet (UV) light. In the present study, we study on the physiological and biochemical response and differentially expressed proteome under UV light irradiation stress in H. armigera adults. Main results were summarized as follows:
1. Response of the antioxidant system of H. armigera adults to UV light stress
We test the hypothesis that one of the effects of UV light irradiation is to increase oxidative stress on insects. The effects of UV light irradiation on total antioxidant capacity and the activities of superoxide dismutase (SOD), catalase (CAT), peroxidases (POX), glutathione-S-transferase (GST) and glutathione reductase (GR) were investigated in H. armigera adults. The adults were exposed to UV light for various time periods (0,30,60 and 90 min). We found that exposure to UV light for 30 min resulted in increased total antioxidant capacity and the activities of SOD, CAT, POX and GST. When the exposure time lasted for 60 and 90 min, the activities of CAT and GST remained significantly higher than the control. However, the antioxidant capacity and SOD activity returned to control levels, and POX activity decreased at 60 and 90 min. Our results confirm the hypothesis that UV light irradiation increases the level of oxidative stress in H. armigera adults.
2. Effects of UV light stress on the activities of acetylcholinesterase and carboxylesterase in H. armigera adults
We analyse the relation between UV light stress and the activities of acetylcholinesterase (AChE) and carboxylesterase (CarE) in H. armigera adults. The effects of UV light irradiation on the activities AChE and CarE were investigated in H. armigera adults. The adults were exposed to UV light for various time periods (0,30, 60 and 90 min). AChE activity of adults exposed to UV light was reduced in comparison with the control adults, and a significant decrease was found at 30 min of exposure. We found decreaded CarE activity in H. armigera adults exposed to UV light for 30,60 and 90 min in comparison with the control. We assume that the activities of AChE and CarE in insects may be influenced by other environmental stress, except for insecticides that have been reported.
3. Effects of UV light stress on several isozymes in H. armigera adults
To study the effects of UV light stress on esterase, POX, and CAT isozymes in H. armigera adults, we carried out isozyme electrophoresis. When exposed to UV light irradiation, esterase isozyme changed mainly in the number and activity of isozyme. At 30 and 60 min exposure, the intensity of isozyme bands E4, E9 and E10 were enhanced, E2 and E8 were decreased. The bands El, E5, E7 and Ell disappered after UV light irradiation, E3 and E6 were new patterns. At the longest exposure time (90 min), the intensity of isozyme bands E4 and E9 E10 were enhanced, E2 and E8 were decreased. The bands E1, E5 and E7 disappered after UV light irradiation, E3 and E6 were new patterns. H. armigera adults showed 6 POX bands. The intensity of POX band P5 was enhanced in adults following exposure to UV light for 30,60 and 90 min. H. armigera adults showed 2 CAT bands. The intensity of band C1 was enhanced in adults following exposure to UV light irradiation for 30,60 and 90 min. The intensity of band C2 was decreased at 30 and 90 min of exposure in comparison with the control. These processes may be mirrored in insect physiological adaptation.
4. A proteomic analysis of H. armigera adults after exposure to UV light irradiation
To gain a better understanding of the response of H. armigera adults to UV light irradiation, we carried out a comparative proteomic analysis. Three-day-old adults were exposed to UV light for 1 h. Total proteins were extracted and separated by two-dimensional gel electrophoresis. More than 1200 protein spots were reproducibly detected, including 12 that were more abundant and 21 less abundant. Mass spectrometry analysis and database searching helped us to identify 29 differentially abundant proteins, including mitogen-activated protein kinase, ste20-like kinase CG40293-PA, isoform A, ADP-ribosylation factor guanine nucleotide-exchange factor 2 (brefeldin A-inhibited), isoform CRA_a, zinc finger protein 768, PREDICTED:similar to PPAR-alpha interacting complex protein 285, Beta subunit of type I geranylgeranyl transferase, putative octanoyltransferase, mitochondrial, heat shock protein hsp21.4, PREDICTED:similar to catalase, enolase, H+ transporting ATP synthase beta subunit isoform 1, H+ transporting ATP synthase beta subunit isoform 2, Prenylcysteine oxidase 1, Adenosine deaminase-like protein A, 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl-pyrophosphate synthetase, actin (Fragment), myosin heavy chain, muscle myosin heavy chain, olfactory receptor-like receptor, lysozyme C, structural maintenance of chromosome, putative, etc. The identified proteins were categorized into several functional groups including signal transduction, RNA processing, protein processing, stress response, metabolisms, and cytoskeleton structure, etc.
1.棉铃虫抗氧化系统对UV胁迫的响应
为了验证UV照射会对昆虫造成氧化胁迫这一假设,本研究检测了UV照射不同时间(0、30、60和90min)对棉铃虫总抗氧化能力的影响；通过测定过氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POX)、谷胱甘肽-S-转移酶(GST)及谷胱甘肽还原酶(GR)活性,分析了棉铃虫体内酶促抗氧化系统对UV胁迫的应答反应。当UV照射棉铃虫成虫30 min时,其体内总抗氧化能力、SOD、CAT、POX和GST活性均显著升高。当照射时间延长至60和90 min时,CAT和GST活性仍显著高于对照组水平,但总抗氧化能力和SOD活性恢复到对照组水平,POX活性则明显降低。本试验证实了UV照射能够对棉铃虫造成氧化胁迫的假设,提出UV照射对趋光性昆虫是一种光胁迫的理论。
2.UV胁迫对棉铃虫乙酰胆碱酯酶和羧酸酯酶活性的影响
本试验探讨了UV胁迫与棉铃虫体内乙酰胆碱酯酶(AChE)及羧酸酯酶(CarE)活性之间的关系,分析比较了两种酶活性在UV照射不同时间(0、30、60和90 min)下的变化情况。棉铃虫经过UV照射不同时间后,体内的AChE活性均有所降低,且在30 min时达到显著水平。UV照射对棉铃虫体内的CarE活性也产生了一定的抑制作用,与对照组相比,照射时间为30、60和90min的UV处理组CarE活性均显著低于对照水平。本研究推测,昆虫体内的AChE和CarE活性除了受已报道的杀虫剂的影响外,也有可能随着其它胁迫环境的作用而发生改变,如UV胁迫。
3.UV胁迫对棉铃虫几种同工酶的影响
为了分析UV胁迫条件下棉铃虫体内酯酶同工酶、POX同工酶和CAT同工酶的变化,本研究采用了同工酶电泳方法。与对照相比,UV处理组酯酶同工酶谱带发生了质和量的变化。照射时间为30和60 min时,谱带E4、E9、E10增强,谱带E2、E8减弱,谱带E1、E5、E7、E11消失,新增了E3、E6谱带；照射时间延长至90 min时,谱带E4、E9增强,谱带E2、E8减弱,谱带E1、E5、E7消失,新增了E3、E6谱带。棉铃虫POX同工酶主要有6条谱带,与对照组相比,棉铃虫P5谱带在UV照射处理下增强。棉铃虫CAT同工酶主要有2条谱带,在经过UV照射不同时间后谱带变化有所不同,与对照组相比,UV处理组的P1谱带增强,而P2谱带在30 min时减弱,60 min时恢复到对照水平,90 min时与对照相比又有所减弱。本研究推测这些同工酶谱带发生的变化,可能是昆虫对UV胁迫条件的一种适应反应。
4.棉铃虫响应UV胁迫的蛋白质组研究
为了研究UV胁迫条件下棉铃虫体内蛋白的变化,本文采用了比较蛋白质组学的方法。分别提取正常条件和UV照射1h条件下的棉铃虫体内总蛋白,然后进行双向电泳分离虫体的总蛋白,共检测到至少1200个蛋白点,其中21个被下调,12个被上调。质谱分析共鉴定出29个蛋白点,包括丝裂原活化蛋白激酶、Ste20类激酶CG40293-PA亚型A、ADP-核糖基化因子鸟苷酸转换因子2(BFA抑制)亚型CRA a、锌指蛋白768、预测的PPAR-α相互作用复合蛋白285相似蛋白、牦牛儿基转移酶Ⅰβ亚基、推测的线粒体辛酰基转移酶、热激蛋白21.4、预测的CAT相似蛋白、烯醇酶、H转运ATP合成酶β亚基亚型1、H转运ATP合成酶β亚基亚型2、异戊烯基半胱氨酸氧化酶1、腺苷脱氨酶相似蛋白A、3-羟-3-甲基戊二酰辅酶A还原酶、法呢基焦磷酸合成酶、肌动蛋白、肌球蛋白重链、肌肉肌球蛋白重链、嗅觉受体类似物、溶菌酶C、推测的染色体结构维持蛋白和一些功能未知的假设蛋白。这些蛋白多数具有重要的生物学功能,参与了信号转导、RNA加工、蛋白质加工、氧化还原平衡、代谢、及细胞骨架稳定等过程。
The cotton bollworm, Helicoverpa armigera (Hubner) (Lepidoptera:Noctuidae) is one of the most serious insect pest in China and neighboring contries. The moths of this nocturnal insect display a conspicuous positive phototactic behaviour to light stimuli, and are especially sensitive to Ultraviolet (UV) light. In the present study, we study on the physiological and biochemical response and differentially expressed proteome under UV light irradiation stress in H. armigera adults. Main results were summarized as follows:
1. Response of the antioxidant system of H. armigera adults to UV light stress
We test the hypothesis that one of the effects of UV light irradiation is to increase oxidative stress on insects. The effects of UV light irradiation on total antioxidant capacity and the activities of superoxide dismutase (SOD), catalase (CAT), peroxidases (POX), glutathione-S-transferase (GST) and glutathione reductase (GR) were investigated in H. armigera adults. The adults were exposed to UV light for various time periods (0,30,60 and 90 min). We found that exposure to UV light for 30 min resulted in increased total antioxidant capacity and the activities of SOD, CAT, POX and GST. When the exposure time lasted for 60 and 90 min, the activities of CAT and GST remained significantly higher than the control. However, the antioxidant capacity and SOD activity returned to control levels, and POX activity decreased at 60 and 90 min. Our results confirm the hypothesis that UV light irradiation increases the level of oxidative stress in H. armigera adults.
2. Effects of UV light stress on the activities of acetylcholinesterase and carboxylesterase in H. armigera adults
We analyse the relation between UV light stress and the activities of acetylcholinesterase (AChE) and carboxylesterase (CarE) in H. armigera adults. The effects of UV light irradiation on the activities AChE and CarE were investigated in H. armigera adults. The adults were exposed to UV light for various time periods (0,30, 60 and 90 min). AChE activity of adults exposed to UV light was reduced in comparison with the control adults, and a significant decrease was found at 30 min of exposure. We found decreaded CarE activity in H. armigera adults exposed to UV light for 30,60 and 90 min in comparison with the control. We assume that the activities of AChE and CarE in insects may be influenced by other environmental stress, except for insecticides that have been reported.
3. Effects of UV light stress on several isozymes in H. armigera adults
To study the effects of UV light stress on esterase, POX, and CAT isozymes in H. armigera adults, we carried out isozyme electrophoresis. When exposed to UV light irradiation, esterase isozyme changed mainly in the number and activity of isozyme. At 30 and 60 min exposure, the intensity of isozyme bands E4, E9 and E10 were enhanced, E2 and E8 were decreased. The bands El, E5, E7 and Ell disappered after UV light irradiation, E3 and E6 were new patterns. At the longest exposure time (90 min), the intensity of isozyme bands E4 and E9 E10 were enhanced, E2 and E8 were decreased. The bands E1, E5 and E7 disappered after UV light irradiation, E3 and E6 were new patterns. H. armigera adults showed 6 POX bands. The intensity of POX band P5 was enhanced in adults following exposure to UV light for 30,60 and 90 min. H. armigera adults showed 2 CAT bands. The intensity of band C1 was enhanced in adults following exposure to UV light irradiation for 30,60 and 90 min. The intensity of band C2 was decreased at 30 and 90 min of exposure in comparison with the control. These processes may be mirrored in insect physiological adaptation.
4. A proteomic analysis of H. armigera adults after exposure to UV light irradiation
To gain a better understanding of the response of H. armigera adults to UV light irradiation, we carried out a comparative proteomic analysis. Three-day-old adults were exposed to UV light for 1 h. Total proteins were extracted and separated by two-dimensional gel electrophoresis. More than 1200 protein spots were reproducibly detected, including 12 that were more abundant and 21 less abundant. Mass spectrometry analysis and database searching helped us to identify 29 differentially abundant proteins, including mitogen-activated protein kinase, ste20-like kinase CG40293-PA, isoform A, ADP-ribosylation factor guanine nucleotide-exchange factor 2 (brefeldin A-inhibited), isoform CRA_a, zinc finger protein 768, PREDICTED:similar to PPAR-alpha interacting complex protein 285, Beta subunit of type I geranylgeranyl transferase, putative octanoyltransferase, mitochondrial, heat shock protein hsp21.4, PREDICTED:similar to catalase, enolase, H+ transporting ATP synthase beta subunit isoform 1, H+ transporting ATP synthase beta subunit isoform 2, Prenylcysteine oxidase 1, Adenosine deaminase-like protein A, 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl-pyrophosphate synthetase, actin (Fragment), myosin heavy chain, muscle myosin heavy chain, olfactory receptor-like receptor, lysozyme C, structural maintenance of chromosome, putative, etc. The identified proteins were categorized into several functional groups including signal transduction, RNA processing, protein processing, stress response, metabolisms, and cytoskeleton structure, etc.
引文
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