白木香细胞色素P450基因AsCYP71D1的克隆及表达分析  

Cloning and expression analysis of cytochrome P450 AsCYP71D1 from Aquilaria sinensis(Lour.)Gilg

作　　者：高世玺 戎梅 彭俊祥 徐艳红 魏建和 GAO Shi-xi;RONG Mei;PENG Jun-xiang;XU Yan-hong;WEI Jian-he(Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine,Ministry of Education&National Engineering Laboratory for Breeding of Endangered Medicinal Materials,Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100193,China;Hainan Provincial Key Laboratory of Resources Conservation and Development of Southern Medicine&Key Laboratory of State Administration of Traditional Chinese Medicine for Agarwood Sustainable Utilization,Hainan Branch of the Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences and Peking Union Medical College,Haikou 57031l,China)

机构地区：[1]中国医学科学院,北京协和医学院,药用植物研究所,中草药物质基础与资源利用教育部重点实验室,濒危药材繁育国家工程实验室,北京100193 [2]中国医学科学院,北京协和医学院,药用植物研究所海南分所,海南省南药资源保护与开发重点实验室,国家中医药管理局沉香可持续利用重点研究室,海南海口570311

出　　处：《药学学报》2023年第10期3123-3129,共7页Acta Pharmaceutica Sinica

基　　金：北京市自然基金项目(7222286);国家自然科学基金项目(81573525);中国医学科学院医学与健康科技创新工程--重大协同创新项目(2021-1-12M-032)。

摘　　要：细胞色素P450(cytochrome P450 monooxygenases,CYP450)是一类超基因家族编码的含有血红素的氧化酶类,分布于各种需氧生物体内,广泛参与萜类、生物碱、黄酮、脂肪酸等的生物合成。本研究依据课题组前期测得白木香转录组数据库中一个CYP450单加氧酶基因的部分转录本序列设计引物,利用逆转录-聚合酶链反应(reverse transcription-PCR,RT-PCR)和cDNA末端快速扩增(rapid-amplification of cDNA ends,RACE)技术克隆该基因的全长cDNA序列;并对其组织表达及亚细胞定位进行了研究。结果发现,该基因全长cDNA序列为1920 bp,其中5′-非翻译区(untranslated region,UTR)为88 bp,3′-UTR为344 bp并具有21 bp的polyA尾,开放阅读框为1488 bp,编码495个氨基酸。序列比对发现该蛋白属于CYP450家族的CYP71D家族成员,命名为AsCYP71D1。组织表达分析的结果显示,AsCYP71D1基因主要在茎中表达。进一步洋葱表皮中亚细胞定位显示,AsCYP71D1编码的蛋白在细胞质、细胞核及细胞膜上都有表达。本研究为后续深入研究其在沉香倍半萜合成途径的功能奠定了基础。Cytochrome P450(CYP450)is a kind of superfamily oxidase containing heme,which is distributed in various aerobic organisms.They are widely involved in the biosynthesis of terpenoids,alkaloids,flavonoids,fatty acids,etc.In this study,the full-length cDNA sequence of a P450 was cloned by reverse transcription-PCR(RT-PCR)and rapid amplification of cDNA ends(RACE)technology,with the specific primers that designed according to the sequence of a transcript annotated as P450 from the Aquilaria sinensis(Lour.)Gilg transcriptome database.The tissue expression and subcellular localization were also studied.The full-length cDNA of the cloned P450 gene is 1920 bp,with 88 bp 5′-untranslated region(UTR),344 bp 3′-UTR and a 21 bp polyA tail,and 1488 bp open reading frame(ORF),encoding 495 amino acids.Sequence alignment revealed that the protein belonged to CYP71D family of cytochrome P450 family,and named AsCYP71D1.Tissue expression analysis indicated that AsCYP7IDI was mainly expressed in stem.Further subcellular localization of onion epidermis showed that AsCYP7IDI was expressed in cytoplasm,nucleus and cell membrane.This study will provide a foundation for further research on its function in agarwood sesquiterpene biosynthesis.

关 键 词：白木香 倍半萜 细胞色素P450 基因克隆 表达分析 

分 类 号：R917[医药卫生—药物分析学]