药学学报, 2014, 49(12): 1724-1729
引用本文:
梁良, 郭庆梅, 张争, 徐艳红, 韩晓敏, 刘娟. 白木香倍半萜合酶基因AsSS4的克隆、原核表达与功能鉴定[J]. 药学学报, 2014, 49(12): 1724-1729.
LIANG Liang, GUO Qing-mei, ZHANG Zheng, XU Yan-hong, HAN Xiao-min, LIU Juan. Cloning, prokaryotic expression, and functional identification of a sesquiterpene synthase gene (AsSS4) from Aquilaria sinensis [J]. Acta Pharmaceutica Sinica, 2014, 49(12): 1724-1729.

白木香倍半萜合酶基因AsSS4的克隆、原核表达与功能鉴定
梁良1,2, 郭庆梅3, 张争1,4, 徐艳红1, 刘娟1
1. 中国医学科学院、北京协和医学院药用植物研究所, 北京 100193;
2. 聊城市人民医院, 山东 聊城 252000;
3. 山东中医药大学药学院, 山东 济南 250012;
4. 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), 海南 万宁 571533;
5. 燕山大学环境与化学工程学院, 河北 秦皇岛 066004
摘要:
通过RT-PCR方法从伤害处理的白木香树木质部中克隆得到沉香倍半萜合酶4 (sesquiterpene synthase 4, AsSS4) 基因的全长开放读码框, 该基因全长读码框为1 698 bp, 编码包含565个氨基酸的蛋白质.将AsSS4基因与原核表达载体pET28a相连构建重组载体pET28a-AsSS4, 把重组载体转入大肠杆菌BL21 (DE3) pLysS中, 用IPTG诱导重组菌, 经SDS-PAGE电泳分析, 获得分子质量约为64 kD的重组AsSS4蛋白.利用镍凝胶亲和色谱法获得纯度较高的AsSS4蛋白, 以法尼基焦磷酸 (FPP) 为底物进行蛋白酶促反应, 共催化产生5种倍半萜类成分: cyclohexane, 1-ethenyl-1-methyl-2, 4-bis(1-methylethenyl)-、β-elemene、α-guaiene、α-caryophyllene和δ-guaiene.本研究首次证实了白木香倍半萜合酶AsSS4基因的功能, 为揭示伤害诱导沉香倍半萜形成的分子机制奠定了理论基础.
关键词:    白木香      倍半萜合酶基因      原核表达      基因功能     
Cloning, prokaryotic expression, and functional identification of a sesquiterpene synthase gene (AsSS4) from Aquilaria sinensis
LIANG Liang1,2, GUO Qing-mei3, ZHANG Zheng1,4, XU Yan-hong1, LIU Juan1
1. Instituent of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China;
2. Liaocheng City People's Hospital, Liaocheng 252000, China;
3. College of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan 250012, China;
4. Hainan Branch of Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Hainan Provincial Key Laboratory of Resource Conservation and Development of Southern Medicine, Wanning 571533, China;
5. School of Environmental and Chemical Engineering, Yan Shan University, Qinhuangdao 066004, China
Abstract:
A sesquiterpene synthase (AsSS4) full-length open reading frame (ORF) cDNA was cloned from wounded stems of Aquilaria sinensis by RT-PCR method. The result showed that the ORF of AsSS4 was 1 698 bp encoding 565 amino acids. Prokaryotic expression vector pET28a-AsSS4 was constructed and transformed into E. coli BL21 (DE3) pLysS. Recombinant AsSS4 protein was obtained after induction by IPTG and SDS-PAGE analysis with a MW of 64 kD. Enzymatic reactions using farnesyl pyrophosphate showed that recombinant AsSS4 protein purified by Ni-agarose gel yielded five sesquiterpene compounds, cyclohexane, 1-ethenyl-1-methyl-2, 4-bis(1-methylethenyl)-, β-elemene, α-guaiene, α-caryophyllene and δ-guaiene. This paper reported the first cloning and functional characterization of AsSS4 gene from A. sinensis, which will establish a foundation for future studies on the molecular mechanisms of wound-induce agarwood formation in A. sinensis.
Key words:    Aquilaria sinensis    sesquiterpene synthase gene    prokaryotic expression    gene function   
收稿日期: 2014-05-19
基金项目: 国家自然科学基金资助项目(31000136,81173481);北京市自然科学基金资助项目(6102024);高等学校博士学科点专项科研基金资助项目(20091106120009);"十二五"国家科技支撑计划项目(2011BAI01B07);海南省中药现代化专项项目(2010ZY001,2012ZY002).
通讯作者: 张争
Email: zhangzheng321@126.com
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