Cloning and function analyses of HDS gene from Artemisia annua / 中草药

ABSTRACT

Objective To obtain the indispensable key enzyme-hydroxide methyl enylamino 4-cyclodiphosphate synthase (HDS) gene involved in the MEP pathway cloned from Artemisia annua and conduct bioinformatic and functional complementation analysis. Methods To perform multiple sequence alignment for the nucleotide acid sequence of the other reported seed plants' HDS gene, to select conservative areas for designing degenerate primers, and to gain the aim gene from A. annua through homologous expanding and cDNA bottom speedily expanding technique. To perform sequence alignment using BLAST, to identify open reading frame (ORF) using ORF Finder, and to construct phylogenetic tree using neighbor joining (NJ) ways in MEGA3.0. Results The obtained HDS cDNA sequence was 2 324 bp containing a 1 854 bp ORF and encoding a 617-amino acid protein. Bioinformatic analysis showed that AaHDS was homologous with HDS derived from other seed plant species. Functional complementation analysis indicated that AaHDS could make up the short HDS function of mutant Escherichia coli MG1655 araHDS. It could make the mutant get back to upgrowth, which showed AaHDS had typical HDS gene function. Conclusion The cloning HDS gene from A. annua for the first time provides a good basis for further study on the metabolization project of artemisinin.