桔小实蝇P450基因CYP6A41的克隆与表达分析  

Cloning and expression analysis of CYP6A41 in Bactrocera dorsalis(Hendel)(Diptera:Tephritidae)

作　　者：胡黎明[1] 申建梅[1] 胡美英[2] 宾淑英[1] 廖泓之[1] 林进添[1] 

机构地区：[1]仲恺农业工程学院农学院,广东广州510225 [2]华南农业大学天然农药与化学生物学教育部重点实验室,广东广州510642

出　　处：《西北农林科技大学学报（自然科学版）》2012年第8期113-121,共9页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(31171852)

摘　　要：【目的】克隆桔小实蝇Bactrocera dorsalis(Hendel)P450基因,研究其在不同组织及发育时期的表达情况,探索其可能存在的生理功能。【方法】采用反转录聚合酶链式反应(RT-PCR)和快速扩增cDNA末端(RACE)技术,克隆桔小实蝇P450基因CYP6A41;采用实时荧光定量PCR(real-time PCR)方法,研究CYP6A41mRNA在桔小实蝇不同组织及不同发育时期的相对表达量。采用Homology程序模拟构建CYP6A41蛋白的三维结构,应用CDOCK程序将之与甲酸乙酯、乙酸乙酯、甲基丁香酚3种气味分子进行模拟对接分析。【结果】克隆获得了桔小实蝇P450基因,并命名为CYP6A41。CYP6A41阅读框全长1 530bp,编码509个氨基酸,该蛋白序列与桔小实蝇的另外一种P450蛋白CYP4 D46序列的一致性最高,达99.2%。荧光定量PCR分析表明,CYP6A41在桔小实蝇不同发育时期都有表达,并且在化蛹第1天表达量达到最高峰;CYP6A41在各组织中也都有表达,但以触角中的表达量最高;雄虫生殖节中的表达量约是雌虫的6.8倍;对接结果表明,CYP6A41蛋白与3种小分子化合物均能形成稳定的复合物。【结论】CYP6A41在桔小实蝇雌雄生殖节中的差异表达,暗示该蛋白在雄虫生殖生理过程中发挥作用;CYP6A41在化蛹第1天及触角中的高表达量暗示CYP6A41不仅参与了蛹早期的发育过程,且可能参与了嗅觉气味分子的降解过程。【Objective】 The Bactrocera dorsalis P450 gene was cloned to determine its expression pattern in all tissues and development stages to explore its possible physiological function.【Method】 Rapid amplification cDNA ends（RACE） method and reverse transcription PCR（RT-PCR） method were used to clone P450 gene.The temporal and spatial expression levels of P450 mRNA were investigated by real-time quantitative RT-PCR method.Homology program was used to build simulated 3D structure of P450;CDOCK program was invited to docking analysis between simulated P450 and three odor molecule,acid ethyl ester,ethyl acetate and Methy leugenol.【Result】 The whole sequence of P450 was gained and named as CYP6A41.The full-length of open reading frame in CYP6A41 is 1 530 bp in length and the putative amino acid shared the highest similarity（99.2%） with CYP4D46,another P450 protein form Bactrocera dorsalis.Real-time PCR results indicated that CYP6A41 expressed in all tissues while the highest expression level was detected in female and male antenna.The amount of CYP6A41 mRNA in male genital segment was about 6.8 folds of that in female counterpart.CYP6A41 mRNA was also detected in all life cycle and the highest expression level in the 1 d-old pupae.Docking result revealed that stable complexes were formed between the simulated CYP6A41 and three compounds,respectively.【Conclusion】Apparently sex-biased expression pattern in the genital segmental implied that CYP6A41 participated in the male reproductive physiological process;Higher expression level in 1 d-old pupae and antenna suggested that CYP6A41 involved not only in the pupa early metamorphosis process but also in the degradation of odour compounds.

关 键 词：桔小实蝇 P450 基因克隆 实时荧光定量PCR 蛋白同源建模 分子对接 

分 类 号：Q785[生物学—分子生物学] Q786