农药与紫外胁迫对白蛾周氏啮小蜂小分子热激蛋白sHSP12.2基因表达的影响
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摘要
【目的】研究寄生性天敌昆虫热激蛋白HSPs的功能,为探讨寄生性天敌昆虫的小分子热激蛋白对农药与紫外胁迫下的抗逆性作用提供基础。【方法】克隆白蛾周氏啮小蜂的一个小热激蛋白基因Ccshsp12.2,对其从系统发育、核苷酸替换速率和密码子使用偏好性等方面进行分析,并利用实时荧光定量PCR(RT-qPCR)技术研究其经紫外照射及用阿维菌素、苏云金杆菌、灭幼脲和高效氯氰菊酯4种农药处理后,Ccshsp12.2的表达量变化。【结果】获得了含有完整编码区的Ccshsp12.2基因序列,编码206个氨基酸,分子量为23.1 kDa;进化分析结果表明白蛾周氏啮小蜂等寄生性类群与麦茎蜂等自由生活类群聚成2个分支。选取的9条基因平均Ka/Ks(即dN/dS)值为0.906 556,主要接受负选择压力。寄生性昆虫的平均密码子选择偏好性指数为0.344,自由生活昆虫的平均密码子选择偏好性指数为0.353。RT-qPCR试验结果显示,与对照相比,经农药和紫外线的处理后,白蛾周氏啮小蜂Ccshsp12.2表达量均有所上调,达3.36～6.17倍。【结论】通过分子进化分析,发现不同生活方式的昆虫其热激蛋白所受的进化压力有显著差别;紫外照射及阿维菌素等4种农药均能显著诱导Ccshsp12.2基因表达上调。本研究结果将为白蛾周氏啮小蜂HSPs的抗逆性研究提供依据。
【Objective】 This study aimed to study the role of small heat shock protein(sHSP) in parasitoids and explore the resistance of the heat shock proteins from parasitic natural enemies to pesticides and ultraviolet stress. 【Method】 The small heat shock protein gene of Chouioia cunea(Ccshsp12.2) was cloned and was used for constructing a phylogenetic tree, calculating amino acid substitution rate and codon usage bias. The RT-qPCR technique was used to quantify the relative expression of Ccshsp12.2 under ultraviolet and treatments with four kinds of pesticides(Abamectin, Bacillus thuringiensis, chlorbenzuron and Beta-cypermethrin).【Result】The Ccshsp12.2 gene with complete ORF was obtained, encoding 206 amino acid with a molecular weight of 23.1 kDa. The phylogenetic trees showed two lineages, Ccshsp12.2 was clustered with other parasitoids in to one group, while Cephus cinctus was clustered with other independent Hymenoptera insects into the other. The average value of Ka/Ks(dN/dS) in 9 selected mRNA sequences is 0.674 067, which means that those genes appear to be primarily negatively selected. In addition, the average codon usage bias of parasite insect is 0.344, while it is 0.353 among independent insect. The result of RT-qPCR showed that Ccshsp12.2 was up-regulated by the effect of insecticides and ultraviolet independently. The relative expression of Ccshsp12.2 was 3.36-6.17 times higher than the control under the treatment of insecticides and ultraviolet A radiation. 【Conclusion】 The phylogenic result and molecular evolution analysis showed that Hymenoptera insects with different living style might suffer from different evolution stress. Ccshsp12.2 was induced in response to ultraviolet and 4 kinds of pesticides. The findings in this study would provide some clues for the investigation about the response of C. cunea to a biotic stresses and insight into the use of this parasitoid in biological control strategies.
【Objective】 This study aimed to study the role of small heat shock protein(sHSP) in parasitoids and explore the resistance of the heat shock proteins from parasitic natural enemies to pesticides and ultraviolet stress. 【Method】 The small heat shock protein gene of Chouioia cunea(Ccshsp12.2) was cloned and was used for constructing a phylogenetic tree, calculating amino acid substitution rate and codon usage bias. The RT-qPCR technique was used to quantify the relative expression of Ccshsp12.2 under ultraviolet and treatments with four kinds of pesticides(Abamectin, Bacillus thuringiensis, chlorbenzuron and Beta-cypermethrin).【Result】The Ccshsp12.2 gene with complete ORF was obtained, encoding 206 amino acid with a molecular weight of 23.1 kDa. The phylogenetic trees showed two lineages, Ccshsp12.2 was clustered with other parasitoids in to one group, while Cephus cinctus was clustered with other independent Hymenoptera insects into the other. The average value of Ka/Ks(dN/dS) in 9 selected mRNA sequences is 0.674 067, which means that those genes appear to be primarily negatively selected. In addition, the average codon usage bias of parasite insect is 0.344, while it is 0.353 among independent insect. The result of RT-qPCR showed that Ccshsp12.2 was up-regulated by the effect of insecticides and ultraviolet independently. The relative expression of Ccshsp12.2 was 3.36-6.17 times higher than the control under the treatment of insecticides and ultraviolet A radiation. 【Conclusion】 The phylogenic result and molecular evolution analysis showed that Hymenoptera insects with different living style might suffer from different evolution stress. Ccshsp12.2 was induced in response to ultraviolet and 4 kinds of pesticides. The findings in this study would provide some clues for the investigation about the response of C. cunea to a biotic stresses and insight into the use of this parasitoid in biological control strategies.
引文
郭虹霞,王创云,赵丽,等 2013. 小分子热激蛋白的研究进展. 山西农业科学, 41(12): 1421-1423.(Guo H X, Wang C Y, Zhao L, et al. 2013. Research progress of small heat shock protein. Journal of Shanxi Agricultural Sciences, 41(12): 1421-1423. [in Chinese])
刘康,王一. 2005. α晶状体蛋白的研究进展. 眼科新进展, 25(2): 171-173.(Liu K, Wang Y. 2005. Progression of alpha crystalline protein. Recent Advances in Ophthalmlogy, 25(2): 171-173. [in Chinese])
莫建初,滕立,杨天赐,等. 2002. 阿维菌素对黄胸散白蚁 Reticulitermes—speratus 的毒效. 农药学学报, 4(4): 86-89.(Mo J C, Teng L, Yang T C, et al. 2002. The toxicity of Abamectin for Reticulitermes speratus. Chinese Journal of Pesticide Science, 4(4): 86-89. [in Chinese])
乔鲁芹,曲良建,杨忠岐,等. 2009. 不同药剂对美国白蛾重要天敌―白蛾周氏啮小蜂的影响. 林业科学研究, 22(4): 559-562.(Qiao L Q, Qu L J, Yang Z Q, et al. 2009. Effects of different pesticides on Chouioia cunea Yang, an important natural enemy of Hyphantria cunea Drury. Forest Research, 22(4): 559-562. [in Chinese])
唐良德,邱宝利,任顺祥. 2014. 天敌昆虫抗药性研究进展. 应用昆虫学报, 51(1): 13-25.(Tang L D, Qiu B L, Ren S X, 2014. A review of insecticide resistance in the natural enemies of pest insects. Chinese Journal of Applied Entomology, 51(1): 13-25. [in Chinese])
王国强. 2010. 小分子热休克蛋白生物学功能的研究进展. 安徽农业科学, 38(19): 9946-9947, 9949.(Wang G Q. 2010. Research progress in biology function of small heat shock proteins. Journal of Anhui Agricultural Science, 38(19): 9946-9947, 9949. [in Chinese])
王洪魁,许国庆 1997. 利用柞蚕蛹人工繁殖白蛾周氏啮小蜂的研究. 沈阳农业大学学报, 28(1): 16-21.(Wang H K, Xu G Q. 1997. Artificial breeding of Chouioia cunea Yang with Tussah pupa. Journal of Shengyang Agricultureal University, 28(1): 16-21. [in Chinese])
肖达,郭晓军,王甦,等 2014. 3种杀虫剂对几种昆虫天敌的毒力测定. 环境昆虫学报, 36(6): 951-958.(Xiao D, Guo X J, Wang S, et al. 2014. The toxicity of three insecticides to natural enemy. Journal of Environment Entomology, 36(6): 951-958. [in Chinese])
杨忠岐. 1989.中国寄生于美国白蛾的啮小蜂一新属一新种(膜翅目,姬小蜂科,啮小蜂亚科),昆虫分类学报,1(1):117-130.(Yang Z Q.1989.A new genus and species of Eulophidae(Hymenoptera:Chalcidoidea)parasitizing Hyphantria cunea (Drury)(Lepidoptera:Arctiidae) in China.Entomotaxonomia,1(1):117-130.[in Chinese])
杨忠岐. 1990. 美国白蛾的有效天敌——白蛾周氏啮小蜂. 森林病虫通讯, (2):7.(Yang Z Q. 1990. The efficient natural enemy of Hyphantria cunea(Drury)- Chouioia cunea Yang. Forest Pest and Disease, (2):7. [in Chinese])
张彦龙,陈合志,武三安 2008. 灭幼脲 Ⅲ 号对白蛾周氏啮小蜂的安全性测试. 安徽农业科学, 36(4): 1486-1487.(Zhang Y L, Chen H Z, Wu S A. 2008. Safety testing of Dimilin III on Chouioia cunea. Journal of Anhui Agricultural Sciences, 36(4): 1486-1487. [in Chinese)
Ali A, Rashid M A, Huang Q Y, et al. 2017. Influence of UV-A radiation on oxidative stress and antioxidant enzymes in Mythimna separata (Lepidoptera: Noctuidae). Environmental Science and Pollution Research, 24(9): 8392-8398.
Chen X E, Zhang Y l. 2015. Identification and characterisation of multiple glutathione S-transferase genes from the diamondback moth, Plutella xylostella. Pest Management Science, 71(4): 592-600.
Colinet H, Renault D, Charoy-Guével B, et al. 2012. Metabolic and proteomic profiling of diapause in the aphid parasitoid Praon volucre. PLoS One, 7(2): e32606.
Crow J F, Kimura M. 1970. An introduction to population genetics theory. First edition. West Caldwell:Blackburn Press, 386-387.
Denlinger D L, Hallman G J, 1998. Temperature sensitivity in insects and application in integrated pest management. Entomologia Experimentalis et Applicata, 91(2): 373-374
Don R, Cox P, Wainwright B, et al. 1991. ‘Touchdown’ PCR to circumvent spurious priming during gene amplification. Nucleic Acids Research, 19(14): 4008.
Feng H, Wang L, Liu Y, et al. 2010. Molecular characterization and expression of a heat shock protein gene (HSP90) from the carmine spider mite, Tetranychus cinnabarinus (Boisduval). Journal of Insect Science, 10(1): 112.
Kim K K, Kim R, Kim S H. 1998. Crystal structure of a small heat-shock protein. Nature, 394(6693): 595-599.
Li Y, Zhang J, Zhao Y, et al. 2014. Characteristic fingerprint based on low polar constituents for discrimination of Wolfiporia extensa according to geographical origin using UV spectroscopy and chemometrics methods. Journal of Analytical Methods in Chemistry, 2014:51924.
Meng J Y, Zhang C Y, Zhu F, et al. 2009. Ultraviolet light-induced oxidative stress: effects on antioxidant response of Helicoverpa armigera adults. Journal of Insect Physiology, 55(6): 588-592.
Nei M, Gojobori T. 1986. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Molecular Biology and Evolution, 3(5): 418-426.
Nguyen T T A, Boudreault S, Michaud D, et al. 2008. Proteomes of the aphid Macrosiphum euphorbiae in its resistance and susceptibility responses to differently compatible parasitoids. Insect Biochemistry and Molecular Biology, 38(7): 730-739.
Rozas J, Sánchez-DelBarrio J C, Messeguer X, et al. 2003. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics, 19(18): 2496-2497.
Sang W, Ma W H, Qiu L, et al. 2012. The involvement of heat shock protein and cytochrome P450 genes in response to UV-A exposure in the beetle Tribolium castaneum. Journal of Insect Physiology, 58(6): 830-836.
Sharp P M, Li W H 1989. On the rate of DNA sequence evolution in Drosophila. Journal of Molecular Evolution, 28(5): 398-402.
Shields D C, Sharp P M, Higgins D G, et al. 1988. “Silent” sites in Drosophila genes are not neutral: evidence of selection among synonymous codons. Molecular Biology and Evolution, 5(6): 704-716.
Stamler R, Kappé G, Boelens W, et al. 2005. Wrapping the α-crystallin domain fold in a chaperone assembly. Journal of Molecular Biology, 353(1): 68-79.
Sun Y, Sheng Y, Bai L, et al. 2014. Characterizing heat shock protein 90 gene of Apolygus lucorum (Meyer-Dür) and its expression in response to different temperature and pesticide stresses. Cell Stress and Chaperones, 19(5): 725-739.
Suzuki Y, Gojobori T. 1999. A method for detecting positive selection at single amino acid sites. Molecular Biology and Evolution, 16(10): 1315-1328.
Tang T, Wu C, Li J, et al. 2012. Stress-induced HSP70 from Musca domestica plays a functionally significant role in the immune system. Journal of Insect Physiology, 58(9): 1226-1234.
Tian H, Yu S, Liu B, et al. 2015. Molecular cloning of heat shock protein gene HSP90 and effects of abamectin and double-stranded RNA on its expression in Panonychus citri (Trombidiformes: Tetranychidae). Florida Entomologist, 98(1): 37-43.
Xu J, Shu J, Zhang Q. 2010. Expression of the Tribolium castaneum (Coleoptera: Tenebrionidae) hsp83 gene and its relation to oogenesis during ovarian maturation. Journal of Genetics and Genomics, 37(8): 513-522.
Yang Z Q, Wei J, Wang X Y. 2006. Mass rearing and augmentative releases of the native parasitoid Chouioia cunea for biological control of the introduced fall webworm Hyphantria cunea in China. Biocontrol, 51(4): 401-418.
Zhang C Y, Meng J Y, Wang X P, et al. 2011. Effects of UV‐A exposures on longevity and reproduction in Helicoverpa armigera, and on the development of its F1 generation. Insect Science, 18(6): 697-702.
Zhao L, Jones W. 2012. Expression of heat shock protein genes in insect stress responses. Invertebrate Surviv J, 9(1):93-101.
Zhu G, Pan L, Zhao Y, et al. 2017. Chemical investigations of volatile kairomones produced by Hyphantria cunea (Drury), a host of the parasitoid Chouioia cunea Yang. Bulletin of Entomological Research, 107(2): 234-240.
刘康,王一. 2005. α晶状体蛋白的研究进展. 眼科新进展, 25(2): 171-173.(Liu K, Wang Y. 2005. Progression of alpha crystalline protein. Recent Advances in Ophthalmlogy, 25(2): 171-173. [in Chinese])
莫建初,滕立,杨天赐,等. 2002. 阿维菌素对黄胸散白蚁 Reticulitermes—speratus 的毒效. 农药学学报, 4(4): 86-89.(Mo J C, Teng L, Yang T C, et al. 2002. The toxicity of Abamectin for Reticulitermes speratus. Chinese Journal of Pesticide Science, 4(4): 86-89. [in Chinese])
乔鲁芹,曲良建,杨忠岐,等. 2009. 不同药剂对美国白蛾重要天敌―白蛾周氏啮小蜂的影响. 林业科学研究, 22(4): 559-562.(Qiao L Q, Qu L J, Yang Z Q, et al. 2009. Effects of different pesticides on Chouioia cunea Yang, an important natural enemy of Hyphantria cunea Drury. Forest Research, 22(4): 559-562. [in Chinese])
唐良德,邱宝利,任顺祥. 2014. 天敌昆虫抗药性研究进展. 应用昆虫学报, 51(1): 13-25.(Tang L D, Qiu B L, Ren S X, 2014. A review of insecticide resistance in the natural enemies of pest insects. Chinese Journal of Applied Entomology, 51(1): 13-25. [in Chinese])
王国强. 2010. 小分子热休克蛋白生物学功能的研究进展. 安徽农业科学, 38(19): 9946-9947, 9949.(Wang G Q. 2010. Research progress in biology function of small heat shock proteins. Journal of Anhui Agricultural Science, 38(19): 9946-9947, 9949. [in Chinese])
王洪魁,许国庆 1997. 利用柞蚕蛹人工繁殖白蛾周氏啮小蜂的研究. 沈阳农业大学学报, 28(1): 16-21.(Wang H K, Xu G Q. 1997. Artificial breeding of Chouioia cunea Yang with Tussah pupa. Journal of Shengyang Agricultureal University, 28(1): 16-21. [in Chinese])
肖达,郭晓军,王甦,等 2014. 3种杀虫剂对几种昆虫天敌的毒力测定. 环境昆虫学报, 36(6): 951-958.(Xiao D, Guo X J, Wang S, et al. 2014. The toxicity of three insecticides to natural enemy. Journal of Environment Entomology, 36(6): 951-958. [in Chinese])
杨忠岐. 1989.中国寄生于美国白蛾的啮小蜂一新属一新种(膜翅目,姬小蜂科,啮小蜂亚科),昆虫分类学报,1(1):117-130.(Yang Z Q.1989.A new genus and species of Eulophidae(Hymenoptera:Chalcidoidea)parasitizing Hyphantria cunea (Drury)(Lepidoptera:Arctiidae) in China.Entomotaxonomia,1(1):117-130.[in Chinese])
杨忠岐. 1990. 美国白蛾的有效天敌——白蛾周氏啮小蜂. 森林病虫通讯, (2):7.(Yang Z Q. 1990. The efficient natural enemy of Hyphantria cunea(Drury)- Chouioia cunea Yang. Forest Pest and Disease, (2):7. [in Chinese])
张彦龙,陈合志,武三安 2008. 灭幼脲 Ⅲ 号对白蛾周氏啮小蜂的安全性测试. 安徽农业科学, 36(4): 1486-1487.(Zhang Y L, Chen H Z, Wu S A. 2008. Safety testing of Dimilin III on Chouioia cunea. Journal of Anhui Agricultural Sciences, 36(4): 1486-1487. [in Chinese)
Ali A, Rashid M A, Huang Q Y, et al. 2017. Influence of UV-A radiation on oxidative stress and antioxidant enzymes in Mythimna separata (Lepidoptera: Noctuidae). Environmental Science and Pollution Research, 24(9): 8392-8398.
Chen X E, Zhang Y l. 2015. Identification and characterisation of multiple glutathione S-transferase genes from the diamondback moth, Plutella xylostella. Pest Management Science, 71(4): 592-600.
Colinet H, Renault D, Charoy-Guével B, et al. 2012. Metabolic and proteomic profiling of diapause in the aphid parasitoid Praon volucre. PLoS One, 7(2): e32606.
Crow J F, Kimura M. 1970. An introduction to population genetics theory. First edition. West Caldwell:Blackburn Press, 386-387.
Denlinger D L, Hallman G J, 1998. Temperature sensitivity in insects and application in integrated pest management. Entomologia Experimentalis et Applicata, 91(2): 373-374
Don R, Cox P, Wainwright B, et al. 1991. ‘Touchdown’ PCR to circumvent spurious priming during gene amplification. Nucleic Acids Research, 19(14): 4008.
Feng H, Wang L, Liu Y, et al. 2010. Molecular characterization and expression of a heat shock protein gene (HSP90) from the carmine spider mite, Tetranychus cinnabarinus (Boisduval). Journal of Insect Science, 10(1): 112.
Kim K K, Kim R, Kim S H. 1998. Crystal structure of a small heat-shock protein. Nature, 394(6693): 595-599.
Li Y, Zhang J, Zhao Y, et al. 2014. Characteristic fingerprint based on low polar constituents for discrimination of Wolfiporia extensa according to geographical origin using UV spectroscopy and chemometrics methods. Journal of Analytical Methods in Chemistry, 2014:51924.
Meng J Y, Zhang C Y, Zhu F, et al. 2009. Ultraviolet light-induced oxidative stress: effects on antioxidant response of Helicoverpa armigera adults. Journal of Insect Physiology, 55(6): 588-592.
Nei M, Gojobori T. 1986. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Molecular Biology and Evolution, 3(5): 418-426.
Nguyen T T A, Boudreault S, Michaud D, et al. 2008. Proteomes of the aphid Macrosiphum euphorbiae in its resistance and susceptibility responses to differently compatible parasitoids. Insect Biochemistry and Molecular Biology, 38(7): 730-739.
Rozas J, Sánchez-DelBarrio J C, Messeguer X, et al. 2003. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics, 19(18): 2496-2497.
Sang W, Ma W H, Qiu L, et al. 2012. The involvement of heat shock protein and cytochrome P450 genes in response to UV-A exposure in the beetle Tribolium castaneum. Journal of Insect Physiology, 58(6): 830-836.
Sharp P M, Li W H 1989. On the rate of DNA sequence evolution in Drosophila. Journal of Molecular Evolution, 28(5): 398-402.
Shields D C, Sharp P M, Higgins D G, et al. 1988. “Silent” sites in Drosophila genes are not neutral: evidence of selection among synonymous codons. Molecular Biology and Evolution, 5(6): 704-716.
Stamler R, Kappé G, Boelens W, et al. 2005. Wrapping the α-crystallin domain fold in a chaperone assembly. Journal of Molecular Biology, 353(1): 68-79.
Sun Y, Sheng Y, Bai L, et al. 2014. Characterizing heat shock protein 90 gene of Apolygus lucorum (Meyer-Dür) and its expression in response to different temperature and pesticide stresses. Cell Stress and Chaperones, 19(5): 725-739.
Suzuki Y, Gojobori T. 1999. A method for detecting positive selection at single amino acid sites. Molecular Biology and Evolution, 16(10): 1315-1328.
Tang T, Wu C, Li J, et al. 2012. Stress-induced HSP70 from Musca domestica plays a functionally significant role in the immune system. Journal of Insect Physiology, 58(9): 1226-1234.
Tian H, Yu S, Liu B, et al. 2015. Molecular cloning of heat shock protein gene HSP90 and effects of abamectin and double-stranded RNA on its expression in Panonychus citri (Trombidiformes: Tetranychidae). Florida Entomologist, 98(1): 37-43.
Xu J, Shu J, Zhang Q. 2010. Expression of the Tribolium castaneum (Coleoptera: Tenebrionidae) hsp83 gene and its relation to oogenesis during ovarian maturation. Journal of Genetics and Genomics, 37(8): 513-522.
Yang Z Q, Wei J, Wang X Y. 2006. Mass rearing and augmentative releases of the native parasitoid Chouioia cunea for biological control of the introduced fall webworm Hyphantria cunea in China. Biocontrol, 51(4): 401-418.
Zhang C Y, Meng J Y, Wang X P, et al. 2011. Effects of UV‐A exposures on longevity and reproduction in Helicoverpa armigera, and on the development of its F1 generation. Insect Science, 18(6): 697-702.
Zhao L, Jones W. 2012. Expression of heat shock protein genes in insect stress responses. Invertebrate Surviv J, 9(1):93-101.
Zhu G, Pan L, Zhao Y, et al. 2017. Chemical investigations of volatile kairomones produced by Hyphantria cunea (Drury), a host of the parasitoid Chouioia cunea Yang. Bulletin of Entomological Research, 107(2): 234-240.
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