刺五加GAPDH基因克隆和序列分析.PDFVIP

中草药 Chinese Traditional and Herbal Drugs 第 43 卷 第 1 期 2012 年 1 月 • 155 • 刺五加 GAPDH 基因的克隆及序列分析 * 邢朝斌 ，吴 鹏，陈 龙，梁能松，何 闪 河北联合大学生命科学学院，河北 唐山 063000 摘 要：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。 关键词：刺五加；GAPDH 基因；克隆；序列分析；内参照基因 中图分类号：R282.12 文献标志码：A 文章编号：0253 - 2670(2012)01 - 0155 - 04 Cloning and sequence analyses of Eleutherococcus senticosus GAPDH XING Zhao-bin, WU Peng, CHEN Long, LIANG Neng-song, HE Shan College of Life Science, Hebei United University, Tangshan 063000, China Abstract: 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 en