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.
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.
Citation: 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

Cloning, prokaryotic expression, and functional identification of a sesquiterpene synthase gene (AsSS4) from Aquilaria sinensis

  • 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.

     

    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.

     

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