| 摘要: |
| 【目的】克隆并鉴定金纹细蛾（Phyllonorycter ringoniella）细胞色素CYP6家族基因，并观察氯氟氰菊酯药剂处理后mRNA相对表达量的变化，为金纹细蛾细胞色素CYP6家族基因全长cDNA的获得，以及防止或延缓金纹细蛾抗药性的产生提供理论依据。【方法】以GenBank上公布的CYP6家族基因编码区序列为基础设计并合成简并引物；利用RT-PCR扩增获得序列后在NCBI上进行Blastx对比，根据P450基因命名法进行命名并向GenBank提交序列获得登录号；利用点滴法和qRT-PCR技术，将金纹细蛾3龄幼虫在氯氟氰菊酯（LC50=12.88 mg/L）条件下处理24 h后，观察其体内细胞色素P450基因的相对表达量。【结果】成功扩增出3个长度分别为415，412和421 bp的cDNA片段。同源性分析表明，3条基因序列与CYP6AN5、CYP6B5和CYP6ab氨基酸相似性分别达61%，59%和71%，可将其初步均归类为CYP6家族，并分别命名为phCYP6AN5、phCYP6B5和phCYP6ab，将序列信息提交至GenBank，获得GenBank登录号分别为KJ559411、KJ559412和KJ559413。药剂处理对3种基因发生了不同的诱导或抑制作用，phCYP6AN5基因在受到药剂处理后相对表达量显著上升，而phCYP6B5和phCYP6ab在受到药剂处理后相对表达量显著下降。【结论】成功获得3条金纹细蛾细胞色素CYP6家族基因片段；诱导剂的诱导作用存在一定程度的特异性，对某种或几种P450酶有效应的化合物并不一定诱导其他类型的P450。 |
| 关键词: 金纹细蛾 细胞色素P450 序列分析 基因表达 |
| DOI： |
| 分类号: |
| 基金项目:陕西省农业攻关项目（2008K01-04）；世界银行资助项目（Y/SHYL/XY/050） |
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| Cloning and expression of cytochrome CYP6 gene from Phyllonorycter ringoniella |
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TIAN Tian,LI Xin,GUO Chang-ning,et al
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| Abstract: |
| 【Objective】This study cloned and identified cytochrome CYP6 gene from Phyllonorycter ringoniella,and observed the change of mRNA relative expression after cyhalothrin treatment to provide information for obtaining full length cDNA of CYP6 gene and preventing or delaying its drug resistance.【Method】Degenerate primers were designed and synthesized based on the available coding region of CYP6 family genes in GenBank database.Blastx comparison on NCBI was conducted after getting the fragments by RT-PCR method,and they were named according to P450 gene nomenclature before being submitted to GenBank for registration number.At last,the relative expression of cytochrome P450 genes was quantified after the 3rd instars of Phyllonorycter ringoniella were disposed in cyhalothrin（LC50=12.88 mg/L）for 24 hours by spot method and qRT-PCR.【Result】Three cDNA fragments with lengths of 415,412,and 421 bp were successfully obtained,and the similarities of amino acids were 61%,59% and 71%,respectively by homology analysis.They were named phCYP6AN5,phCYP6B5 and phCYP6ab and preliminarily grouped to CYP6 family.The login numbers of KJ559411,KJ559412 and KJ559413 were obtained from GenBank.The expression of phCYP6AN5 after treatment increased,while that of phCYP6B5 and phCYP6ab decreased.【Conclusion】This study successfully constructed three cytochrome CYP6 family genes and they had specificity to inducing agents.The compounds that were effective to P450 enzyme may not necessarily work for other types of P450. |
| Key words: Phyllonorycter ringoniella cytochrome P450 sequence analysis gene expression |
