刺五加亚精胺合成酶基因的克隆及内生真菌对其表达的影响

Cloning of spermidine synthase gene in Eleutherococcus senticosus and effect of endophytic fungus on its expression

目的克隆刺五加亚精胺合成酶(spermidine synthase,SPDS)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SPDS基因全长cDNA序列。运用生物信息学方法对该基因进行分析。RT-PCR法检测内生真菌菌株P116-1a、P116-1b、P109-4和P312-1对SPDS基因表达的影响。结果刺五加SPDS基因的cDNA全长为1 541 bp,开放阅读框长1 002 bp,编码333个氨基酸的蛋白,包含SPDS家族的基本结构和标志性序列。RT-PCR结果显示,内生真菌可显著提高刺五加SPDS基因的表达量(P<0.05),最大表达量出现在菌株P116-1b回接90 d时,是对照的2.06倍。结论首次克隆了刺五加SPDS基因的cDNA全长序列,并证实内生真菌可显著提高刺五加SPDS基因的表达,为阐明内生真菌提高刺五加三萜皂苷量的机制及刺五加的抗逆性改良奠定了基础。

Objective In order to clone spermidine synthase （SPDS） gene in Eleutherococcus senticosus and analyze the effects of endophytic fungi on its expression. Methods The SPDS full-length cDNA sequence of E. senticosus was cloned by rapid amplification of cDNA ends （RACE）. The gene was analyzed by the bioinformatics method. The effects of endophytic fungi, P 116-1 a, P 116-1 b, P 109-4, and P312-1, on SPDS expression were detected by RT-PCR. Results The full-length cDNA of E. senticosus SPD S gene was 1 541 bp containing an open reading frame length of 1 002 bp that encoded protein with 333 amino acids. The predicted protein included the basic structure and typical sequences of SPDS family. RT-PCR results showed that endophytic fungi could significantly improve SPDS gene expression amount （P 〈 0.05）. The highest expression amount of SPDS showed up on day 90 after reinoculation with P116-1b, which was as much as 2.06 times of the control. Conclusion The full-length cDNA sequence of E. senticosus SPDS gene is successfully cloned and reported for the first time. The results demonstrate that endophytic fungi could obviously improve SPDS gene expression. This result could provide a foundation for clarifying the mechanism that endophytic fungi could improve the content of triterpenoid saponins in E. senticosus and for stressing the tolerance improvement.