药学学报, 2017, 52(1): 172-179
引用本文:
李笑, 强玮, 邱飞, 陈敏, 兰小中, 廖志华, 刘小强. 天仙子中参与托品烷生物碱生物合成的ArAT基因的克隆及功能鉴定[J]. 药学学报, 2017, 52(1): 172-179.
LI Xiao, QIANG Wei, QIU Fei, CHEN Min, LAN Xiao-zhong, LIAO Zhi-hua, LIU Xiao-qiang. Cloning and characterization of an aromatic amino acid aminotransferase (ArAT) gene involved in tropane alkaloid biosynthesis from Hyoscyamus niger[J]. Acta Pharmaceutica Sinica, 2017, 52(1): 172-179.

天仙子中参与托品烷生物碱生物合成的ArAT基因的克隆及功能鉴定
李笑1,2, 强玮1,2, 邱飞1,2, 陈敏3, 兰小中4, 廖志华1,2, 刘小强1,2
1. 西南大学生命科学学院, 三峡库区教育部生态环境教育部重点实验室, 重庆 400715;
2. 西南大学-西藏农牧学院药用植物联合研发中心, 重庆 400715;
3. 西南大学药学学院, 重庆 400715;
4. 西藏大学农牧学院, 西藏 850000
摘要:
托品烷类生物碱是临床上广泛应用的抗胆碱药物,其生物合成涉及到苯丙氨酸的转氨基反应。根据天仙子(Hyoscyamus niger)侧根和叶的转录组数据,筛选到3条功能注释为芳香族氨基酸氨基转移酶的基因,命名为HnArAT1、HnArAT2HnArAT3。通过序列同源性分析,发现HnArAT3与颠茄的AbArAT4的氨基酸序列同源性最高,两者为直系同源。组织表达谱显示,HnArAT3基因在根中特异性表达,与其他3个合成途径基因(PMT、TRIH6H)表达模式相同。用病毒诱导的基因沉默(VIGS)方法在天仙子植株中鉴定HnArAT3的功能,对感染了病毒的植株,用实时荧光定量PCR检测基因的表达量,HPLC法测定托品烷类生物碱的含量。结果表明,在pTRV2-HnArAT3干扰的植株中,HnArAT3基因的表达量有显著下降,3种托品烷类生物碱莨菪碱、山莨菪碱和东莨菪碱含量也有显著的降低。以上结果表明HnArAT3是天仙子中参与托品烷类生物碱生物合成的苯乳酸支路途径基因。HnArAT3基因的克隆为进一步研究托品烷类生物碱的生物合成和代谢调控奠定了基础,也为开展托品烷类生物碱的代谢工程研究提供了新的候选靶基因。
关键词:    天仙子      芳香族氨基酸氨基转移酶      病毒诱导的基因沉默      托品烷类生物碱     
Cloning and characterization of an aromatic amino acid aminotransferase (ArAT) gene involved in tropane alkaloid biosynthesis from Hyoscyamus niger
LI Xiao1,2, QIANG Wei1,2, QIU Fei1,2, CHEN Min3, LAN Xiao-zhong4, LIAO Zhi-hua1,2, LIU Xiao-qiang1,2
1. Key Laboratory of Eco-environments in Three Gorges Reservoir Region, Ministry of Education of PRC, School of Life Sciences, Southwest University, Chongqing 400715, China;
2. SWU-TAAHC Medicinal Plant Joint R & D Centre, Chongqing 400715, China;
3. School of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China;
4. Agricultural and Husbandry College, Tibet University, Tibet 850000, China
Abstract:
Tropane alkaloids are anticholinergic drugs widely used clinically. Biosynthesis of tropane alkaloids in planta involves a step of transamination of phenylalanine. Based on the sequenced transcriptomes of lateral roots and leaves of Hyoscyamus niger, we found three annotated aromatic amino acid aminotransferases, which were respectively named HnArAT1, HnArAT2 and HnArAT3. Sequence analysis showed that HnArAT3 had highest similarity with the reported Atropa belladonna AbArAT4, which was involved in tropane alkaloid (TA) to provide the precursor of the phenyllactic acid moiety. Tissue expression pattern analysis indicated that HnArAT3 was specifically expressed in lateral roots, where is the organ synthesizing tropane alkaloids. Then, method of virus induced gene silencing (VIGS) was used to characterize the function of HnArAT3 in H. niger. Gene expression analysis given by real-time quantitative PCR showed that all the transgenic lines had lower expression levels of HnArAT3 than the non-transgenic control, and HPLC analysis of alkaloids demonstrated significant decrease in the contents of hyoscyamine, anisodamine and scopolamine in planta. These results suggested that HnArAT3 was involved in the phenyllactic acid branch of TA biosynthetic pathway. Molecular cloning and functional identification of HnArAT3 laid the foundation for further understanding of TA biosynthesis and metabolic regulation, and also provided a new candidate gene for engineering biosynthetic pathway of tropane alkaloids.
Key words:    Hyoscyamus niger    aromatic amino acid aminotransferase    virus induced gene silencing    tropane alkaloids   
收稿日期: 2016-06-21
DOI: 10.16438/j.0513-4870.2016-0601
基金项目: 国家863计划资助项目(2011AA100605);国家自然科学基金资助项目(31370333);教育部新世纪优秀人才计划资助项目(NCET-12-0930);中央高校基本科研业务费专项资助项目(XDJK2013A024);中央高校基本业务费专项资助项目(XDJK2014C064).
通讯作者: 刘小强,Tel/Fax:86-23-68367146,E-mail:liuxq@swu.edu.cn
Email: liuxq@swu.edu.cn
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