艾龙脑脱氢酶基因AArBDH1的克隆及功能鉴定  

Cloning and Functional Verification of the Borneol Dehydrogenase Encoding Gene AArBDH1 in Artemisia argyi

作　　者：陈昌婕 苗玉焕 罗丹丹 王梓欣 郭璐娟 赵婷婷 刘大会[1] Changjie Chen;Yuhuan Miao;Dandan Luo;Zixin Wang;Lujuan Guo;Tingting Zhao;Dahui Liu(Resource Center for Chinese Materia Medica,Hubei University of Chinese Medicine,Wuhan 430065,China)

机构地区：[1]湖北中医药大学中药资源中心,武汉430065

出　　处：《植物学报》2023年第4期560-572,共13页Chinese Bulletin of Botany

基　　金：湖北省自然科学基金(No.2021CFB225);国家中医药管理局青年岐黄学者支持项目;中国博士后科学基金(No.2022M711101);国家重点研发计划(No.2017YFC1700704);湖北省教育厅中青年人才项目(No.Q20212001);现代农业产业技术体系建设专项(No.CARS-21);中央本级重大增减支项目(No.2060302)。

摘　　要：龙脑是艾(Artemisia argyi)叶中最重要的药效成分之一,具有抗菌、消炎及镇痛等药理活性。其生物合成和代谢受多种酶的影响,其中龙脑脱氢酶是将龙脑氧化为樟脑的关键酶之一。利用气相色谱-质谱联用(GC-MS)技术测定了35份艾种质叶片中龙脑和樟脑的含量。结果表明,不同品种艾叶片中龙脑和樟脑含量差异较大,在部分种质中樟脑含量甚至超过了龙脑,说明大量的龙脑被氧化为樟脑后严重降低了艾叶中龙脑的含量。基于全长转录组和同源性比较分析,在艾叶中克隆到1个艾龙脑脱氢酶编码基因AArBDH1。AArBDH1基因含有2个外显子和1个内含子,编码包含289个氨基酸残基的蛋白。qRT-PCR分析表明,在艾不同组织和不同发育时期的叶片中AArBDH1呈差异表达,在茎和30天叶龄的叶片中高表达。以龙脑为底物、NAD+为辅酶的酶促反应表明, AArBDH1能催化龙脑脱氢生成樟脑。该研究为进一步揭示艾叶片中龙脑积累的调控和改进机制提供了理论依据和基因资源。Borneol is one of the most important pharmacodynamic components in Artemisia argyi,which has antibacterial,anti-inflammatory,analgesic and other pharmacological activities.The synthesis and metabolism of borneol are affected by many kinds of enzymes,and borneol dehydrogenase is one of the key enzymes that oxidize borneol to camphor.In this study,the contents of borneol and camphor in 35 germplasm of A.argyi leaves were determined by Gas chromatograph-mass spectrometer(GC-MS).We found that the amount of borneol and camphor was highly varied among the varieties,and some germplasm contained more camphor than borneol,indicating that a large proportion of borneol was oxidized to camphor in A.argyi leaves.Based on the full-length transcriptome of A.argyi and homology comparison analysis,we cloned the first borneol dehydrogenase encoding gene AArBDH1 in A.argyi.The AArBDH1 contained 2 exons and 1 intron,and encoded a protein of 289 amino acids.The AArBDH1 gene expression levels were measured by real-time quantitative reverse transcription PCR(qRT-PCR)technology,which showed that AArBDH1 was differentially expressed in different tissues and in leaves at different development stages,and highly expressed in stem and 30 days old leaves.The enzymatic reactions of AArBDH1 with borneol as substrate and NAD+as coenzyme showed that AArBDH1 could catalyze the dehydrogenation of borneol to camphor.Our study provides a theoretical basis and gene resource for further analyzing the regulation and potential improvement of borneol accumulation in A.argyi leaves.

关 键 词：艾 龙脑 龙脑脱氢酶 樟脑 

分 类 号：Q943.2[生物学—植物学] S567.239[农业科学—中草药栽培]