ABSTRACT
Objective: To clone and analyze the full length DNA and promoter sequence of GAPDH gene from Eleutherococcus senticosus. Methods: PCR and TAIL-PCR techniques were used to clone the full length DNA sequence and promoter sequence of GAPDH gene of E. senticosus, then these two sequences were analyzed by bioinformatics methods. Results: Length of 4 103 bp of E. senticosus GAPDH gene DNA and promoter sequence was cloned. Gene structure analysis showed that it contained 12 exons and 11 introns, and the splicing principles of its exon and intron were consistent with GT-AG; The promoter sequence length was 1 304 bp, and the transcription start site located 61 bp upstream of the initiation codon ATG; The promoter elements such as TATA-box, CAAT-box, as well as many cis-regulatory elements were related to hormone signal response, light response and stress signals. Conclusion: The full length DNA and promoter sequence of GAPDH gene in E. senticosus is successfully cloned and reported for the first time, and it provides a stable foundation for further study of GAPDH gene structure and function of E. senticosus.
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 coined 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 £. 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.