植物次生物质2-十三烷酮能够诱导棉铃虫细胞色素P450 CYP6B6基因的过量表达,以6龄棉铃虫(Helicoverpa armigera)幼虫为对象,研究2-十三烷酮对棉铃虫细胞色素P450 CYP6B6的诱导作用,并对其表达规律进行了初探。分别采用实时荧光定量PCR(qRT-PCR)和免疫组化的方法,分析棉铃虫在终浓度为10 mg·g-1 的2-十三烷酮胁迫下,不同时间及不同组织部位CYP6B6 基因在mRNA水平和蛋白水平上的表达变化规律。qRT-PCR结果显示:用添加了2-十三烷酮的人工饲料饲喂棉铃虫不同时间后,在头壳中CYP6B6 mRNA的表达量一直低于对照组,到48 h时与对照组具有显著差异(t4=15.32,P=0.000 1);在脂肪体中随着时间的推移,表达量呈升高趋势,在36 h 已过量表达(t4=7.627,P=0.001 6);在24 h时体壁和中肠组织的表达量分别较对照组提高了2.06倍和7.16倍,之后表达量均呈现先降低后升高的趋势,且在48 h时达到最大。免疫组化结果显示:在棉铃虫幼虫组织中发现CYP6B6蛋白在体壁、脂肪体、中肠中均有表达,相比之下在体壁中表达量较高。该研究为深入了解棉铃虫P450 CYP6B6的表达调控机制及其参与杀虫剂抗性的研究奠定了基础。
The cytochrome P450s in insect can be induced by pesticides, drugs, chemicals and other inducers. The cytochrome P450 CYP6B6 gene was over-expressed after induced by the secondary plant substance 2-tridecanone, so we investigated the induced function of 2-tridecanone and the expression profile of CYP6B6 in sixth-instar of Helicoverpa armigera. In different tissues and at different time, we detected the CYP6B6 expression from cotton bollworm at mRNA level and protein level by Real-Time Quantitative PCR (qRT-PCR) and immunohistochemistry, respectively. qRT-PCR results showed that, after feeding on the cotton bollworm with 10 mg per artificial diet of 2-tridecanone at different time, the expression level of CYP6B6 gene was lower in the head capsule all the time, and still had a significant difference compared with the controlled group at 48 h (t4=15.32, P=0.000 1). The expression level was increased with the time in the fat body and was over-expressed at 36 h (t4=7.627,P=0.001 6); At 24 h, compared with the controlled group, the expression level in the integumentary and midgut was increased up to 2.06-and 7.16-fold respectively. Then the expression level was decreased firstly and then increased, and both reached the maximum level at 48 h; Immunohistochemistry results revealed that the CYP6B6 was all expression in integumentary, fat body and midgut, moreover the CYP6B6 expression level in integumentary was more than that in other tissues in the 6th larvae. This study provides a molecular foundation for the further studies on the expression regulation of cytochrome P450 CYP6B6 and insecticide resistance of Helicoverpa armigera.
〔1〕 Qiu X H,Li W,Tian Y,et al.Cytochrome P450 monooxygenases in the cotton bollworm (Lepidoptera:Noctuidae):Tissue difference and induction〔J〕.Journal of Economic Entomology,2003,96(4):1 283-1 289.
〔2〕 Liu X N,Liang P,Gao X W,et al.Induction of the cytochrome P450 activity by plant allelochemicals in the cotton bollworm,Helicoverpa armigera(Hübner)〔J〕.Pesticide Biochemistry Physiology,2006,84(2):127-134.
〔3〕 Zhang H,Tang T,Cheng Y,et al.Cloning and expression of cytochrome P450 CYP6B7 in fenvalerate-resistant and susceptible Helicoverpa armigera(Hübner) from China〔J〕.Journal Applied Entomology,2010,134(9/10):754-761.
〔4〕 Ahmad M,Arif M I,Ahmad Z.Resistance to carbamate insecticides in Helicoverpa armigera(Lepidoptera:Noctuidae) in Pakistan〔J〕.Crop Protection,2001,20(5):427-432.
〔5〕 Scott J G.Cytochrome P450 monooxygenase mediated resistance to insecticides〔J〕.Journal Pesticide Science,1996,21(2):241-245.
〔6〕 Scott J G.Insect cytochrome P450s:Thinking beyond detoxification〔J〕.Recent Advances in Insect Physiology,Toxicology and Molecular Biology,2008,661(2):117-124.
〔7〕 Harrison T L,Zangerl A R,Schuler M A,et al.Developmental variation in cytochrome P450 expression in Papilio polyxenes in response to xanthotoxin,a hostplant allelochemicals〔J〕.Archives of Insect Biochemistry Physiology,2001,48(4):179-189.
〔8〕 张昕,任春红,甄天民.昆虫细胞色素P450与抗药性关系的分子生物学研究进展〔J〕.中国寄生虫病防治杂志,2002,15(1):62-64.〔Zhang Xin,Ren Chunhong,Zhen Tianmin.Advanced on molecular biology study of relationship between insect Cytochrome P450 and resistance〔J〕.Chinese Journal Parasitic Disease Control,2002,15(1):62-64.〕
〔9〕 周国理,黄炯烈,詹希美.昆虫CYP6家族与抗药性及其转录调控〔J〕.寄生虫与医学昆虫学报,2000,7(4):247-256.〔Zhou Guoli,Huang Jionglie,Zhan Ximei.CYP6 Family in insects,insecticide resistance and its transcriptional control〔J〕.Acta Parasitologica et Medicine Entomologica Sinica,2000,7(4):247-256.〕
〔10〕 Kimps N W,Bissnger B W,Apperson C S,et al.First report of the repellency of 2-tridecanone against ticks〔J〕.Medical and Veterinary Entomology,2011,25(2):202-208.
〔11〕 Danielson P B,MacIntyre R J,Fogleman J C.Molecular cloning of a family of xenobiotic inducible drosophila cytochrome P450s:Evidence for involvement in host plant allelochemicals resistance〔J〕.Proceedings of the National Acad Sciences of the United States of America,1997,94(20):10 797-10 802.
〔12〕 Li X C,Berenbaum M R,Schuler M A.Molecular cloning and expression of CYP6B8:A xanthotoxin-inducible cytochrome P450 cDNA from Helicoverpa zea〔J〕.Insect Biochemistry and Molecular Biology,2000,30(1):75-84.
〔13〕 Cohen M B,Schuler M A,Berenbaum M R.A host plant inducible cytochrome P450 from a host-specific caterpillar:Molecular cloning and evolution〔J〕.Proceedings of the National Acad Sciences of the United States of America,1992,89(22):10 920-10 924.
〔14〕 Hung C F,Berenbaum M R,Schuler M A.Isolation and characterization of CYP6B4,a furanocoumarin inducible cytochrome P450 from a polyphagous caterpillar(Lepidoptera:Papilionidae)〔J〕.Insect Biochemistry and Molecular Biology,1997,27(5):377-385.
〔15〕 郭亭亭,姜辉,高希武.昆虫细胞色素P450基因的多样性、进化及表达调控〔J〕.昆虫学报,2009,52(3):301-311.〔Guo Tingting,Jiang Hui,Gao Xiwu.Diversity,evolution and expression regulation of cytochrome P450 monooxygenase genes in insects〔J〕.Acta Entomolgica Sinica,2009,52(3):301-311.〕
〔16〕 Fujii-Kuriyama Y,Mimura J.Molecular mechanism of AhR function in the regulation of cytochrome P450 genes〔J〕.Biochemical and Biophysical Research Communications,2005,338(1):311-317.
〔17〕 邱星辉,冷欣夫.昆虫细胞色素P450研究:P450酶系的可诱导性〔J〕.昆虫知识,1999,36(1):48-53.〔Qiu Xinghui,Leng Xinfu.The research of the Insects cytochrome P450:The induced ability of P450 enzyme〔J〕.Entomological Knowledge,1999,36(1):48-53.〕
〔18〕 Terriere L C.Induction of detoxication enzymes in insects〔J〕.Annual Review of Entomology,1984,29:71-88.
〔19〕 Brattsten L B.Ecological significance of mixed-function oxidations〔J〕.Drug Metabolism Reviews,1979,10(1):35-58.
〔20〕 Thongsinthusak T C,Krieger R I.Inhibitory and inductive effects of piperonyl butoxide on dihydroisodrin hydroxylation in vivo and in vitro in black cutworm(Agrotis ypsilon) larvae〔J〕.Life Science,1974,14(11):2 131-2 141.
〔21〕 Brattsten L B,Price S L,Gunderson C A.Microsomal oxidases in midgut and fatbody tissues of a broadly herbivorous insect larva,Spodoptera eridania cramer(Noctuidae)〔J〕.Comparative Biochemistry and Physiology PartC:Comparative Pharmacology,1980,66(2):231-237.
〔22〕 Li X C,Berenbaum M R,Schuler M A.Plant allelochemicals differently regulate Helicoverpa zea cyptochrome P450 genes〔J〕.Insect Molecular Biology,2002,11(4):343-351.
〔23〕 Tate L G,Nakat S S,Hodgson E.Comparison of detoxication activity in midgut and fat body during fifth instar development of the tobacco hornworm,Manduca sexta〔J〕.Comparative Biochemistry and Physiology PartC:Comparative Pharmacology,1982,72(1):75-81.
〔24〕 Dowd P F,Sparks T C.Comparisons of midgut,fat body,and cuticular enzymes from Pseudoplusia includens(Walker) and Heliothis virescens(F.) responsible for the hydrolysis of permethrin and fenvalerate〔J〕.Pesticide Biochemistry and Physiology,1987,27(3):309-317.
〔25〕 何海,刘小宁.新疆有毒植物资源及其植物源杀虫剂开发应用前景〔J〕.干旱区研究,2012,29(1):115-121.〔He Hai,Liu Xiaoning.Prospect of development and utilization of toxic plant resources and their botanical pesticides in Xinjiang〔J〕.Arid Zone Research,2012,29(1):115-121.〕
