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目的克隆获得黄花蒿MEP途径中必需关键酶——羟甲基丁烯基-4-磷酸合成酶基因(HDS),并进行生物信息学分析和功能互补分析研究。方法对已知的其他种子植物HDS基因的核苷酸序列进行多重序列比对,选取保守区域设计简并引物,利用同源扩增和cDNA末端快速扩增技术从黄花蒿中获得目的基因;利用BLAST进行序列比对,ORF Finder寻找开放阅读框,并用MEGA3.0中的临位相联法构建进化树。结果得到1条长2 324 bp的HDS cDNA序列,其ORF框长1 854 bp,编码617个氨基酸残基的蛋白;生物信息学分析显示,黄花蒿HDS基因AaHDS与其他种子植物来源的HDS高度同源;功能互补分析表明,AaHDS能互补突变菌株Escherichia coli MG1655 ara<>HDS中缺失的HDS功能,使突变菌株恢复生长,证明AaHDS具有典型的HDS基因功能。结论首次克隆获得黄花蒿HDS基因,为青蒿素的代谢工程研究提供相应的基础。
Objective To clone and obtain HDS gene, a key enzyme involved in MEP pathway of Artemisia annua L., and perform bioinformatics analysis and functional complementation analysis. Methods The nucleotide sequences of HDS genes from other seed plants were compared using multiple sequence alignment. The conserved regions were designed to design degenerate primers, and the target genes were obtained from A. annua using homologous amplification and rapid amplification of cDNA ends. BLAST sequence alignment, ORF Finder looking for open reading frame, and the use of MEGA3.0 paralogs to build the phylogenetic tree. The results showed that one HDS cDNA sequence with a length of 2 324 bp contained an ORF of 1 854 bp and encoded a protein of 617 amino acid residues. Bioinformatics analysis showed that the HDS gene AaHDS of A. annua was highly homologous with HDS derived from other seed plants The functional complementation analysis showed that AaHDS could complement the mutant HDS function of Escherichia coli MG1655 ara HDS, and restore the growth of mutant strains. It was proved that AaHDS has typical HDS gene function. Conclusion The HDS gene of Artemisia annua L. was cloned for the first time, providing the basis for the metabolic engineering of artemisinin.