摘要
目的对金龙胆草Conyza blinii 3-磷酸甘油醛脱氢酶(GAPDH)基因进行克隆及序列分析,并检测其是否可作为金龙胆草的内参基因。方法采用RACE技术克隆金龙胆草GAPDH基因的cDNA全长序列,利用DNAMAN、BLAST、MEGA等工具对序列进行生物信息学分析,并以其为内参进行半定量RT-PCR。结果克隆获得了1个全长1 418 bp的GAPDH基因,完整开放阅读框1 020 bp,编码340个氨基酸,命名为GLGAPDH。序列比对结果显示,金龙胆草的GAPDH基因与紫背天葵的同源序列相似性达到96%,与薇甘菊的同源序列相似性为93%。通过系统进化树分析发现其与紫背天葵的亲缘关系最近。半定量RT-PCR分析GLGAPDH作为内参基因在不同组织中表达稳定,扩增效果良好,重现性强。结论首次克隆了金龙胆草GAPDH基因的全长cDNA序列,并通过半定量实验验证了其可作为内参基因用于基因表达量分析,为金龙胆草次生代谢物合成过程中关键酶表达分析及调控机制的研究奠定了基础。
Objective To clone and analyze glyceraldehyde-3-phosphate dehydrogenase （GAPDH） gene from Conyza blinii and detect whether it can be the reference gene for C. blinii. Methods Full length GAPDH was cloned by RACE. DNAMAN, BLAST, and MEGA bioinformatic tools were used to analyze its open reading frame （ORF）, homology, and phylogenetic tree. The sequence was acted as internal control gene for semi-quantitative RT-PCR. Results The gene designated GLGAPDH was 1 418 bp in length. It contained a 1 020 bp ORF encoding 340 amino acids. It shared 96% similarity with Gynura bicolor GAPDH and shared 93% similarity with Mikania micrantha GAPDH. GLGAPDH had closer relationship with GAPDH in G. bicolor. When the sequence acted as internal control gene, the semi-quantitative RT-PCR had benign amplification and good reproducibility. Conclusion GAPDH is cloned from C. blinii for the first time. The semi-quantitative RT-PCR results prove that GAPDH gene is able to be the reference gene for gene expression analysis. The result of this study will provide the basis for the key enzyme expression and regulate the mechanism analysis in C. blinii effective components biosynthesis pathway.
出处
《中草药》
CAS
CSCD
北大核心
2013年第19期2732-2735,共4页
Chinese Traditional and Herbal Drugs
基金
四川省科技创新苗子工程资助项目(2012ZZ046)
关键词
金龙胆草
3-磷酸甘油醛脱氢酶基因
克隆
序列分析
内参基因
Conyza blinii L6vl.
glyceraldehyde-3-phosphate dehydrogenase gene
clone
sequence analysis
internal control gene
