铁棒锤转录组分析及乌头碱生物合成相关基因的挖掘  

Transcriptome analysis to identify genes involved in the biosynthesis of aconitines in Aconitum pendulum

作　　者：田梅 陈灵丽 靳保龙 郭娟 葛慧 赵鑫 崔光红[1] TIAN Mei;CHEN Ling-li;JIN Bao-long;GUO Juan;GE Hui;ZHAO Xin;CUI Guang-hong(National Resource Center for Chinese Medicinal Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Gansu University of Traditional Chinese Medicine,Hezheng Medicinal Botanical Garden,Hezheng 731299,China)

机构地区：[1]中国中医科学院中药资源中心,北京100700 [2]甘肃中医药大学和政药用植物园,甘肃和政731299

出　　处：《药学学报》2021年第12期3353-3361,共9页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81891010,81891013);国家重点研究计划资助项目(2018YFA0900600);财政部和农业农村部:国家现代农业产业技术体系资助项目(CARS-21)。

摘　　要：铁棒锤Aconitum pendulum为常用藏药,乌头碱类化合物是其主要活性成分,具有较高的药用价值。为研究铁棒锤乌头碱类生物合成途径,首次采用Illumina HiSeqTM2000高通量测序技术对铁棒锤转录组进行分析,分别构建其根、叶和花转录组数据库。Trinity de novo组装得到47264条unigenes,平均长度1140 bp,N50为1678 bp,30231条unigenes(63.96%)在7大公共数据库得到注释。KEGG分析发现542条unigenes参与17个次生代谢通路,56条unigenes编码乌头碱生物合成20种关键酶基因。其中44条unigenes编码乌头碱类二萜母核生物合成的20种酶类,12个BAHD酰基转移酶基因推测参与乌头碱酰基化修饰,且在不同部位表达存在差异,推测ApTPS8高表达场所(根)是二萜生物碱前体合成的主要部位。结合乌头碱类成分在植物体内的分布规律,推测ApBAHD1/2/8参与2-羟基乌头碱、dehydrated 14-benzoylaconitine、8-O-methyl-14-benzoylaconine、benzoyldeoxyaconitine和benzoylaconitine的生物合成,ApBAHD10参与乌头碱、lucidusculine、14-O-acetylneoline、14-O-acetylvirescenin的生物合成。通过铁棒锤、乌头比较转录组分析,发现铁棒锤中二萜合酶和酰基转移酶基因家族发生明显的收缩,这与乌头碱类化合物种类和数量明显偏少的结果一致,提示铁棒锤为乌头碱类化合物生物合成途径研究的理想材料。本工作可为后续乌头碱生物合成途径解析、毒性形成分子机制的探讨、道地药材形成机制提供基础科学资料。Aconitum pendulum is a Tibetan medicine that is rich in bioactive compounds such as aconitinetype C19-diterpenoid alkaloids.To investigate the key enzymes in the aconitine biosynthesis pathway,roots,leaves and flowers of Aconitum pendulum were subjected to a high-throughput transcriptomic sequencing analysis by Illumina HiSeqTM2000.Trinity de novo assembly yielded 47264 unigenes with an average length of 1140 bp and N50 of 1678 bp,of which 30231 unigenes(63.96%)were annotated.In the KEGG database,542 unigenes were implicated in 17 secondary metabolic pathways;the analysis showed that 44 genes encoded 20 key enzymes in the diterpene skeleton of aconitine biosynthesis and 12 BAHD acyltransferase genes were related to the acetylation modification,with differential expression among three organs.For example,ApTPS8 was the only committed enzyme in the upstream aconitine biosynthetic pathway.The high expression level of ApTPS8 in root indicated that it is the main tissue for the production of precursors of diterpene alkaloids.Consistent with the accumulation of aconitine,we propose that ApBAHD1/2/8 is involved in the biosynthesis of 2-hydroxyaconitine,dehydrated 14-benzoylaconitine,8-O-methyl-14-benzoylaconine,benzoyldeoxyaconitine and benzoylaconitine,and ApBAHD10 is involved in the biosynthesis of acontine,lucidusculine,14-O-acetylneoline and 14-O-acetylvirescenin.Comparative transcriptome analysis of A.pendulum and A.carmichaeli indicates significant gene loss in the family of diterpene synthases and acyltransferases in A.pendulum,which is in accordance with the significantly fewer type and quantity of aconitine compounds in this species.Therefore,A.pendulum has proved to be an ideal material for the study of the aconitine biosynthesis pathway.This work provides basic scientific data for further study of aconitine biosynthesis,the discussion of molecular mechanisms of toxicity,and the synthesis of genuine medicinal materials.

关 键 词：铁棒锤 二萜生物碱 转录组 萜类合酶 酰基转移酶 

分 类 号：R931[医药卫生—生药学]