Cloning and sequence analysis of glyceraldehyde-3-phosphate dehyrogenase gene from Conyza blinii / 中草药

ABSTRACT

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.