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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 from Acanthopanax senticosus and analyze the effect of endophytic fungi on its expression. Methods: The full length cDNA of CaM gene of Acanthopanax senticosus was cloned by rapid amplification of cDNA ends (RACE) technique. The effects of endophytic fungi strains P116-1a, P116-1b, P109-4 and P312-1 on the expression of CaM were detected by RT-PCR. Results: The cDNA of CaM gene was 856 bp in length and 450 bp in open reading frame. It encoded a protein of 149 amino acids with a homology of 100% with those of Panax ginseng and Daucus carota. The results of RT-PCR showed that endophytic fungi could significantly increase the CaM gene expression (P <0.05), and the maximum expression reached 2.96 times of the control when the strain P109-4 was connected 90 days later. CONCLUSION: The full-length cDNA sequence of CaM in Acanthopanax senticosus was cloned and reported for the first time. It was confirmed that endophytic fungi can significantly increase CaM gene expression in Acanthopanax senticosus, which laid the foundation for clarifying the mechanism of endophytic fungi in increasing the content of Acanthopanax senticosus saponins The foundation.