姜黄倍半萜合酶基因ClFLDH的克隆和原核表达分析

Cloning and Prokaryotic Expression Analysis of Sesquiterpene Synthase Gene ClFLDH from Curcuma longa

摘要:

姜黄倍半萜合酶法尼醇脱氢酶基因(ClFLDH)是萜类化合物合成途径中的关键限速酶。为探讨提高姜黄萜烯类化合物产量，该研究通过同源克隆获得ClFLDH的cDNA序列，其开放阅读框(ORF)为855 bp，编码295个氨基酸。生物信息学分析表明，ClFLDH蛋白的相对分子量为31 kDa，等电点为6.79，为脂溶性疏水中性蛋白。构建pCE32a-FLDH原核表达载体，并在大肠杆菌DH5α中进行表达分析，重组菌株的SDS-PAGE电泳检测结果表明目的蛋白大小为37 kDa。GC-MS分析表明，ClFLDH蛋白可能催化3,5-二甲氧基苯酚生成[1,2-a]吡嗪-1,4-二酮,6H-3-(苯甲基)-吡咯。这为深入理解姜黄萜类化合物合成的调控机制提供了新的视角，也为通过基因工程手段提高姜黄萜烯类化合物产量奠定基础。

Abstract:

The farnesol dehydrogenase gene (ClFLDH) of Curcuma longa is a key rate-limiting enzyme in the terpene compound synthesis pathway. To explore the improvement of terpene compound production in C. longa, the cDNA sequence of ClFLDH was obtained through homologous cloning. The open reading frame (ORF) of ClFLDH was 855 bp, encoding 295 amino acids. Bioinformatics analysis indicated that the relative molecular mass of ClFLDH protein was 31 kDa, with an isoelectric point of 6.79, and it was a liposoluble, hydrophobic and neutral protein. The pCE32a-FLDH prokaryotic expression vector was constructed and expressed in Escherichia coli DH5α. The SDS-PAGE electrophoresis results of the recombinant strain showed that the size of the target protein was 37 kDa. GC-MS analysis indicated that the ClFLDH protein might catalyze 3,5-dimethoxyphenol to generate [1,2-a]pyrazine-1,4-dione, 6H-3-(benzyl)-pyrrole. These would provide a new perspective for in-depth understanding of the regulatory mechanism of curcuminoid terpene synthesis and lay a foundation for increasing the production of curcuminoid terpene compounds through genetic engineering.