论文部分内容阅读
青蒿素是治疗疟疾的首选药物,定位于质体的MEP途径可为青蒿素在内的萜类物质的合成提供基本前体。羟甲基丁烯基-4-磷酸还原酶[hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase,HDR]是MEP途径最后一个酶,催化HMBPP生成IPP和IPP的同分异构体DMAPP。本文克隆了黄花蒿HDR基因(Aa HDR2)并进行了相关功能研究。通过对Aa HDR2基因(Gen Bank:KX058541)和已报道的Aa HDR1基因(Gen Bank:ADC84348.1)表达分析结果表明:Aa HDR1和Aa HDR2在黄花蒿腺体中表达量均远高于在根、茎、叶和花中的表达量,而且Aa HDR2在花中的表达量要明显高于叶中;Me JA和ABA能够大幅度上调Aa HDR1和Aa HDR2的表达,而GA3仅能大幅度上调Aa HDR2的表达。据Aa HDR1和Aa HDR2的表达分析表明,Aa HDR2对青蒿素在内的萜类物质的生物合成贡献更大,因此用Aa HDR2进行后续的实验研究。生物信息学分析表明,Aa HDR2属于HDR家族,进一步采用功能互补在E.coli突变体MG1655ara<>HDR中证明Aa HDR2编码蛋白质的确具有HDR的功能。Aa HDR2亚细胞定位结果显示:Aa HDR2融合GFP蛋白特异性定位于叶绿体中,符合MEP途径定位于质体的事实。采用转基因技术在拟南芥中过表达Aa HDR2能显著性提高叶绿素a、叶绿素b和类胡萝卜素含量。因此,本文所克隆的Aa HDR2是利用代谢工程技术提高萜类物质生物合成能力的一个候选基因。
Artemisinin is the drug of choice for malaria. The MEP pathway, located on the plastid, can provide basic precursors for the synthesis of terpenoids, including artemisinin. Hydroxymethyl butenyl 4-phosphate reductase (HDR), the last enzyme in MEP pathway, catalyzes the homologation of IPP and IPP in HMBPP Structure DMAPP. In this paper, HDR gene (Aa HDR2) of A. annua was cloned and related functions were studied. The expression analysis of Aa HDR2 gene (Gen Bank: KX058541) and reported Aa HDR1 gene (Genbank: ADC84348.1) showed that the expression levels of Aa HDR1 and Aa HDR2 in A. annua were much higher than those in roots , Stems, leaves and flowers, and the expression level of Aa HDR2 in flowers was significantly higher than that in leaves. Me JA and ABA could significantly up-regulate the expression of Aa HDR1 and Aa HDR2, while GA3 could only increase significantly Aa HDR2 expression. According to the expression analysis of Aa HDR1 and Aa HDR2, the contribution of Aa HDR2 to the biosynthesis of terpenoids, including artemisinin, is greater, so follow-up experimental studies with Aa HDR2 were performed. Bioinformatics analysis showed that Aa HDR2 belonged to the HDR family and further confirmed that Aa HDR2-encoding protein does have HDR function by complementation of function in E. coli mutant MG1655ara <> HDR. The results of Aa HDR2 subcellular localization showed that the Aa HDR2 fusion GFP protein is localized in the chloroplast, which is consistent with the fact that the MEP pathway is located on the plastid. Overexpression of Aa HDR2 in transgenic Arabidopsis transgenic plants significantly increased chlorophyll a, chlorophyll b and carotenoid content. Therefore, the cloned Aa HDR2 is a candidate gene that uses metabolic engineering to improve the biosynthesis of terpenoids.