甜菜夜蛾细胞色素P450基因的克隆与分析
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摘要
【目的】探索甜菜叶蛾产生抗药性的分子机制。【方法】利用RACE技术,从甜菜夜蛾Spodoptera exigua中克隆获得1个细胞色素P450基因的全长cDNA序列。【结果】该基因全长894 bp,开放阅读框411 bp,3′和5′端非编码序列分别为79和213 bp,推导的氨基酸序列编码194个氨基酸,推测编码蛋白质的分子量为22.30 kDa,等电点为8.22。同源性比对分析发现,甜菜夜蛾细胞色素P450基因与斜纹夜蛾Spodoptera litura、甘蓝夜蛾Mamestra brassicae、棉铃虫Helicoverpa armigera和谷实夜蛾Helicoverpa zea细胞色素P450的相似性分别为42%、41%、39%和42%。【结论】研究结果可为今后深入研究甜菜夜蛾细胞色素P450功能提供借鉴和参考。
【Objective】 The purpose of the article was to study the molecular mechanisms of drug resistance in Spodoptera exigua. 【Method】 The full-length cDNA sequence of cytochrome P450 in Spodoptera exigua was cloned by using RACE technology. 【Result】 The length of this gene was 894 bp with 3 ′and 5′ non-coding sequences of 79 bp and 213 bp. It contained an open reading frame of 411 bp, and encoded 194 amino acids. The protein of the cloned gene had the predicted molecular weight and isoelectric point of 22.30 kDa and 8.22, respectively. Homology analysis found that S. exigua cytochrome P450 showed 42 %, 41 %, 39 % and 42 % similarities to the counterparts of Spodoptera litura, Mamestra brassicae, Helicoverpa armigera and Helicoverpa zea. 【Conclusion】 The results lay the foundation for future in-depth study of S. exigua cytochrome P450.
【Objective】 The purpose of the article was to study the molecular mechanisms of drug resistance in Spodoptera exigua. 【Method】 The full-length cDNA sequence of cytochrome P450 in Spodoptera exigua was cloned by using RACE technology. 【Result】 The length of this gene was 894 bp with 3 ′and 5′ non-coding sequences of 79 bp and 213 bp. It contained an open reading frame of 411 bp, and encoded 194 amino acids. The protein of the cloned gene had the predicted molecular weight and isoelectric point of 22.30 kDa and 8.22, respectively. Homology analysis found that S. exigua cytochrome P450 showed 42 %, 41 %, 39 % and 42 % similarities to the counterparts of Spodoptera litura, Mamestra brassicae, Helicoverpa armigera and Helicoverpa zea. 【Conclusion】 The results lay the foundation for future in-depth study of S. exigua cytochrome P450.
引文
[1]冷欣夫,唐振华,王荫长.农药的分子毒理学和昆虫抗药性[M]. 北京:中国农业出版社, 1996.
[2]苏寿汦,叶炳辉.现代医学昆虫学[M]. 北京:中国高等教育出版社, 1996.
[3]Nelson D R, Kamataki T, Waxman D J, et al. The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature[J]. Dna & Cell Biology, 1993, 12(1):1-51.
[4]Omura T, Gotoh O. Evolutionary Origin of Mitochondrial Cytochrome P450[J]. Journal of Biochemistry, 2017, 161(5):399-407.
[5]蔡双虎,程立生.昆虫细胞色素P-450基因的研究进展[J].热带农业科学, 2002, 22(6):76-82.
[6]Sutherland T D, Unnithan G C, Andersen J F, et al. A cytochrome P450 terpenoid hydroxylase linked to the suppression of insect juvenile hormone synthesis[J]. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(22):12884-12889.
[7]Winter J, Bilbe G, Richener H, et al. Cloning of a cDNA Encoding a Novel Cytochrome P450 from the Insect Locusta migratoria: CYP6H1, a Putative Ecdysone 20-Hydroxylase[J]. Biochemical & Biophysical Research Communications, 1999, 259(2):305-310.
[8]Davies L, Williams D R, Turner P C, et al. Characterization in relation to development of an ecdysteroid agonist-responsive cytochrome P450, CYP18A1, in Lepidoptera[J]. Archives of Biochemistry & Biophysics, 2006, 453(1):4-12.
[9]王玉成,张国栋,姜静.一种适用范围广的总RNA提取方法[J].植物研究,2006, 26(1):84-87.
[10]朱家颖,杨璞,吴国星,等.丽蝇蛹集金小蜂气味结合蛋白基因的克隆与序列分析[J]. 环境昆虫学报, 2010, 32(4):476-482.
[11]罗婵,汤刚彬,谢体三,等.感受态细胞制备与保存方法的比较研究[J]. 生物技术, 2005, 15(1):52-54.
[12]Thompson J D, Gibson T J, Plewniak F, et al. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools[J]. Nucleic Acids Research, 1997, 25(25):4876-4882.
[13]Bendtsen J D, Nielsen H, Heijne G V, et al. Improved Prediction of Signal Peptides: SignalP 3.0[J]. Journal of Molecular Biology, 2004, 340(4):783-795.
[14]Jiang H B, Wang J J, Liu G Y, et al. Molecular Cloning and Sequence Analysis of a Novel P450 Gene Encoding CYP345D3 from the Red Flour Beetle, Tribolium castaneum[J]. Journal of Insect Science, 2008, 8(55):1-7.
[15]Zhou H, Chen K, Yao Q, et al. Molecular cloning of Bombyx mori cytochrome P450 gene and its involvement in fluoride resistance[J]. Journal of Hazardous Materials, 2008, 160(2-3):330-336.
[16]Willoughby L, Batterham P, Daborn P J. Piperonyl butoxide induces the expression of cytochrome P450 and glutathione S-transferase genes in Drosophila melanogaster[J]. Pest Management Science, 2010, 63(8):803-808.
[17]S. Tarès, J B. Bergé, Amichot M. Cloning and Expression of Cytochrome P450 Genes Belonging to the CYP4 Family and to a Novel Family, CYP48, in Two Hymenopteran Insects, Trichogramma cacoeciae and Apis mellifera[J]. Biochemical & Biophysical Research Communications, 2000, 268(3):677-682.
[18]Mao W, Rupasinghe S G, Johnson R M, et al. Quercetin-metabolizing CYP6AS enzymes of the pollinator Apis mellifera, (Hymenoptera: Apidae)[J]. Comparative Biochemistry & Physiology Part B Biochemistry & Molecular Biology, 2009, 154(4):427-434.
[19]Pedra J H, Brandt A, Li H M, et al. Transcriptome identification of putative genes involved in protein catabolism and innate immune response in human body louse (Pediculicidae: Pediculus humanus)[J]. Insect Biochemistry & Molecular Biology, 2003, 33(11):1135-1143.
[20]薛正楷,魏弘.细胞色素P4503A46基因的异源表达及药物代谢活性分析[J]. 西南农业学报, 2015, 28(1):381-385.
[2]苏寿汦,叶炳辉.现代医学昆虫学[M]. 北京:中国高等教育出版社, 1996.
[3]Nelson D R, Kamataki T, Waxman D J, et al. The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature[J]. Dna & Cell Biology, 1993, 12(1):1-51.
[4]Omura T, Gotoh O. Evolutionary Origin of Mitochondrial Cytochrome P450[J]. Journal of Biochemistry, 2017, 161(5):399-407.
[5]蔡双虎,程立生.昆虫细胞色素P-450基因的研究进展[J].热带农业科学, 2002, 22(6):76-82.
[6]Sutherland T D, Unnithan G C, Andersen J F, et al. A cytochrome P450 terpenoid hydroxylase linked to the suppression of insect juvenile hormone synthesis[J]. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(22):12884-12889.
[7]Winter J, Bilbe G, Richener H, et al. Cloning of a cDNA Encoding a Novel Cytochrome P450 from the Insect Locusta migratoria: CYP6H1, a Putative Ecdysone 20-Hydroxylase[J]. Biochemical & Biophysical Research Communications, 1999, 259(2):305-310.
[8]Davies L, Williams D R, Turner P C, et al. Characterization in relation to development of an ecdysteroid agonist-responsive cytochrome P450, CYP18A1, in Lepidoptera[J]. Archives of Biochemistry & Biophysics, 2006, 453(1):4-12.
[9]王玉成,张国栋,姜静.一种适用范围广的总RNA提取方法[J].植物研究,2006, 26(1):84-87.
[10]朱家颖,杨璞,吴国星,等.丽蝇蛹集金小蜂气味结合蛋白基因的克隆与序列分析[J]. 环境昆虫学报, 2010, 32(4):476-482.
[11]罗婵,汤刚彬,谢体三,等.感受态细胞制备与保存方法的比较研究[J]. 生物技术, 2005, 15(1):52-54.
[12]Thompson J D, Gibson T J, Plewniak F, et al. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools[J]. Nucleic Acids Research, 1997, 25(25):4876-4882.
[13]Bendtsen J D, Nielsen H, Heijne G V, et al. Improved Prediction of Signal Peptides: SignalP 3.0[J]. Journal of Molecular Biology, 2004, 340(4):783-795.
[14]Jiang H B, Wang J J, Liu G Y, et al. Molecular Cloning and Sequence Analysis of a Novel P450 Gene Encoding CYP345D3 from the Red Flour Beetle, Tribolium castaneum[J]. Journal of Insect Science, 2008, 8(55):1-7.
[15]Zhou H, Chen K, Yao Q, et al. Molecular cloning of Bombyx mori cytochrome P450 gene and its involvement in fluoride resistance[J]. Journal of Hazardous Materials, 2008, 160(2-3):330-336.
[16]Willoughby L, Batterham P, Daborn P J. Piperonyl butoxide induces the expression of cytochrome P450 and glutathione S-transferase genes in Drosophila melanogaster[J]. Pest Management Science, 2010, 63(8):803-808.
[17]S. Tarès, J B. Bergé, Amichot M. Cloning and Expression of Cytochrome P450 Genes Belonging to the CYP4 Family and to a Novel Family, CYP48, in Two Hymenopteran Insects, Trichogramma cacoeciae and Apis mellifera[J]. Biochemical & Biophysical Research Communications, 2000, 268(3):677-682.
[18]Mao W, Rupasinghe S G, Johnson R M, et al. Quercetin-metabolizing CYP6AS enzymes of the pollinator Apis mellifera, (Hymenoptera: Apidae)[J]. Comparative Biochemistry & Physiology Part B Biochemistry & Molecular Biology, 2009, 154(4):427-434.
[19]Pedra J H, Brandt A, Li H M, et al. Transcriptome identification of putative genes involved in protein catabolism and innate immune response in human body louse (Pediculicidae: Pediculus humanus)[J]. Insect Biochemistry & Molecular Biology, 2003, 33(11):1135-1143.
[20]薛正楷,魏弘.细胞色素P4503A46基因的异源表达及药物代谢活性分析[J]. 西南农业学报, 2015, 28(1):381-385.