刺五加钙调蛋白基因的克隆及内生真菌对其表达的影响

Cloning of Eleutherococcus senticosus calmodulin gene and effect of endophytic fungus on expression amount of gene

目的:克隆刺五加的钙调蛋白(calmodulin,CaM)基因,并分析内生真菌对其表达的影响。方法:采用cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术克隆刺五加CaM基因的全长cDNA序列。通过RT-PCR法检测可显著提高刺五加皂苷含量的内生真菌菌株P116-1a,P116-1b,P109-4,P312-1对CaM表达的影响。结果:刺五加CaM基因的cDNA全长为856 bp,开放阅读框长450 bp,编码149个氨基酸的蛋白,与人参Panax ginseng和胡萝卜Daucus carota等物种的CaM同源性均高达100%。RT-PCR的结果显示,内生真菌可显著提高刺五加CaM基因的表达量(P<0.05),最大表达量出现在菌株P109-4回接90 d时,达对照的2.96倍。结论:首次克隆并报道了刺五加CaM的cDNA全长序列,并证实内生真菌可显著提高刺五加CaM基因的表达量,为阐明内生真菌提高刺五加三萜皂苷含量的机制奠定了基础。

Objective：To clone calmodulin（CaM） gene in Eleutherococcus senticosus,and study the effect of endophytic fungi on expression amount of CaM gene.Method： The CaM full length cDNA sequence was cloned by rapid amplification of cDNA ends（RACE）.The gene was analyzed and corresponding structure and functions were predicted by the bioinformatics methods.The expression amount of CaM gene affected of endophytic fungus P116-1a,P116-1b,P109-4 and P312-1 was detected by RT-PCR.Result： The full length of CaM cDNA was 856 bp containing an ORF of 450 bp that encoded a protein of 149 amino acids.The homologous of predicted protein was almost 100% with plants like Panax ginseng and Daucus carota.RT-PCR results showed that endophytic fungus improved CaM expression amount significantly（P〈0.05）.The highest expression amount of CaM occurred 90 d after reinoculated with endophytic fungi P109-4,up to 2.96 times of the control.Conclusion： The CaM gene of E.senticosus was successfully cloned for the first time.The results demonstrated that endophytic fungus of E.senticosus improved CaM expression amount significantly.