黄花蒿羟甲基丁烯基-4-磷酸还原酶基因克隆与功能研究  

Molecular cloning and functional characterization of the gene encoding hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase gene from Artemisia annua L.

作　　者：曹芳[1,2] 夏菁[1,2] 陈俞裴 张曼[1,2] 向礼恩[1,2] 曾俊岚 陈敏[3] 兰小中[4] 廖志华[1,2] 

机构地区：[1]西南大学生命科学学院三峡库区生态环境教育部重点实验室,重庆400715 [2]西南大学-西藏农牧学院药用植物联合研发中心,重庆400715 [3]西南大学药学院,重庆400715 [4]西藏农牧学院药用植物研究中心,西藏林芝860000

出　　处：《药学学报》2016年第11期1791-1798,共8页Acta Pharmaceutica Sinica

基　　金：国家"863"计划资助项目(2011AA100605);国家自然科学基金资助项目(31070266);教育部新世纪人才支持计划资助项目(NCET-12-0930);西南大学中央高校基本科研业务费专项资助项目(XDJK2013A024)

摘　　要：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 first choice for malaria treatment. The plastidial MEP pathway provides 5-carbon precursors （IPP and its isomer DMAPP） for the biosynthesis of isoprenoid （including artemisinin）.Hydroxy-2-methyl-2-（E）-butenyl 4-diphosphate reductase （HDR） is the last enzyme involved in the MEP pathway, which catalyzes HMBPP to form IPP and DMAPP. In this study, we isolated the full-length cDNA of HDR from Artemisia annua L. （AaHDR2） and performed functional analysis. According to gene expression analysis of AaHDR2 （GenBank： KX058541） and AaHDR1 reported ever （GenBank： ADC84348.1） by qPCR, we found that AaHDR1 and AaHDR2 had much higher expression level in trichomes than that in roots, stems, leaves and flowers. AaHDR2 had much higher expression level in flowers than that in leaves. Further, the plant hormones such as MeJA and ABA respectively up-regulated the expression level of AaHDR1 and AaHDR2 significantly, but GA3 up-regulated the expression level of AaHDR2 only. The gene expression analysis of AaHDR1 and AaHDR2 showed that AaHDR2 had a greater contribution than AaHDR1 to isoprenoid biosynthesis （including artemisinin）. We used AaHDR2 for the following experiments. Bioinformatic analysis indicated that AaHDR2 belonged to the HDR family and the functional complementation assay showed that AaHDR2 did have the enzymatic function of HDR, using E. coli mutant MG1655ara〈〉HDR as host cell. The subcellular localization assay showed that AaHDR2 fused with GFP at its N-terminal specifically targeted in chloroplasts. Finally, AaHDR2 was overexpressed in Arabidopsis thaliana. The AaHDR2-overexpressing plants produced the isoprenoids including chlorophyll a, chlorophyll b and carotenoids at significantly higher levels than the wild-type Arabidopsis plants. In summary, AaHDR2 might be a candidate gene for genetic improvement of the isoprenoid biosynthesis.

关 键 词：黄花蒿 羟甲基丁烯基-4-磷酸还原酶 功能互补 基因表达 亚细胞定位 过表达 

分 类 号：R931[医药卫生—生药学]