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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,As SS4)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As SS4基因与原核表达载体p ET28a相连构建重组载体p ET28a-As SS4,把重组载体转入大肠杆菌BL21(DE3)p Lys S中,用IPTG诱导重组菌,经SDS-PAGE电泳分析,获得分子质量约为64 k D的重组As SS4蛋白。利用镍凝胶亲和色谱法获得纯度较高的As SS4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As SS4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。
The full-length open reading frame of sesquiterpene synthase 4 (As SS4) gene was cloned by RT-PCR from the damaged xylem of Arundinacea wood, and the full-length reading frame of this gene was 1 698 bp, encoding a protein containing 565 amino acids. As SS4 gene was ligated with the prokaryotic expression vector p ET28a to construct the recombinant vector p ET28a-As SS4. The recombinant vector was transformed into E. coli BL21 (DE3) p Lys S and the recombinant strain was induced by IPTG. A recombinant As SS4 protein with a molecular mass of about 64 kD. As SS4 protein with high purity was obtained by Ni-gel affinity chromatography and protease was catalyzed by farnesyl pyrophosphate (FPP). Five kinds of sesquiterpenoids were co-catalyzed: cyclohexane, 1-ethenyl -1-methyl-2,4-bis (1-methylethenyl) -, β-elemene, α-guaiene, α-caryophyllene and δ-guaiene. This study, for the first time, confirmed the function of As SS4 gene of the sesquiterpene synthase, and laid the theoretical foundation for revealing the molecular mechanism of injury-induced sesbania.