刺五加环阿屯醇合酶基因的克隆及其表达分析

Cloning of cycloartenol synthase gene from Eleutherococcus senticosus and its expression analysis

目的克隆刺五加的环阿屯醇合酶(cycloartenol synthase,CAS)基因,并对其进行生物信息学和表达分析。方法采用cDNA末端快速扩增(rapid amplification of cDNAends,RACE)技术克隆刺五加CAS基因的全长cDNA序列。运用生物信息学方法对该基因进行分析,预测其编码蛋白的结构与功能,并通过RT-PCR法检测CAS在不同生长发育时期和不同器官中的表达情况。结果刺五加CAS基因的cDNA全长为2 758 bp,开放阅读框长2 277 bp,编码758个氨基酸的蛋白,包含三萜合成酶的标志性序列。CAS蛋白无跨膜区域,定位于细胞质中。RT-PCR的结果显示,刺五加CAS基因在各时期和器官中均有表达,但表达量具有显著差异(P<0.05)。其中果实基本成熟期的表达量最高,是最低量萌芽期的1.56倍,各器官中,叶片的表达量最高,是最低量叶柄的1.37倍。结论首次分离并报道了刺五加的CAS cDNA克隆,并证实其在不同生长发育时期和不同器官中的表达量不同,为进一步研究CAS对刺五加皂苷量的影响和表达调控奠定基础。

Objective To clone cycloartenol synthase （CAS） gene from Eleutherococcus senticosus and analyze its bioinfonnatics and expression. Methods CAS gene full length eDNA was cloned by rapid amplification of eDNA ends （RACE）. CAS gene was analyzed by bioinformatics method, and the structure and function of CAS gene were deduced. The expression of CAS in different organs of E. senticosus at various growing periods was detected by RT-PCR. Results The full length of CAS gene cDNA was 2 758 bp containing a 2 277 bp open reading frame （ORF） that encoded a protein of 758 amino acids includinng triterpene synthase signature sequence. Without transmembrane domain, CAS gene was located in cytoplasm. RT-PCR result showed that CAS gene expressed in different organs orE. senticosus at various growing periods showed significant difference （P 〈 0.05）. The highest content of the expression showed up when the fiuit almost maturated, which was 1.56 times as much as that in the lowest as plant sprouts. The highest content of the expression was in the leaves which was 1.37 times as much as that of the lowest in petiole. Conclusion The CAS gene orE. senticosus is successfully cloned and reported for the first time which proves that the expression in different organs of E. senticosus at various growing periods is different. The cloning of CAS gene provides a stable foundation for its effect on saponins amount orE. senticosus and expression regulation.