刺五加GAPDH基因的克隆及序列分析

目的 对刺五加Eleutherococcus senticosus GAPDH基因进行克隆及序列分析，使其成为可用的内参照基因。方法 采用改良的异硫氰酸胍法提取刺五加总RNA，运用RT-PCR法克隆GAPDH基因的部分序列，并以其为内参照基因进行半定量PCR。结果 克隆了长度为627 bp的刺五加GAPDH基因，推测其编码209个氨基酸，与三七、人参、拟南芥的GAPDH氨基酸序列同源性分别为97%、93%、93%，核苷酸同源性分别为94%、86%、84%。其作为内参照基因的半定量PCR具有良好的扩增效果和重现性。结论 首次分离并克隆了刺五加GAPDH cDNA，证实该序列可以作为基因表达分析的内参照基因，为刺五加苷生物合成中关键酶的表达分析及调控机制研究奠定基础。

Cloning and sequence analyses of Eleutherococcus senticosus GAPDH

Objective In order to make the gene a valuable internal control gene, GAPDH of Eleutherococcus senticosus was cloned and analyzed in sequence. Methods Total RNA of E. senticosus was extracted using improved isothiocyanate method. Part sequence of GAPDH was colned by RT-PCR, and the sequence was acted as internal control gene for semiquantitative PCR. Results Length 627 bp of E. senticosus GAPDH was cloned, speculating coding 209 amino acids. To compare the amino acid sequence of E. senticosus GAPDH with those of Panax notoginseng, P. ginseng, and Arabidopsis thaliana, the amino acid homology was 97%, 93%, and 93%, respectively, and the nucleotide homology was 94%, 86%, and 84%, respectively. When the sequence acted as internal control gene, the semiquantative PCR has benign amplification effect and good reproducibility. Conclusion The cDNA clone of E. senticosus GAPDH is first reported, the results prove that the sequence is able to be internal control gene for analysis on gene expression. This study could make a foundation for the key enzyme expression and regulate mechanism analysis in eleutheroside biosynthesis pathway.