【作者】 张曼；

【导师】 廖志华；

【作者基本信息】 西南大学 ， 植物学， 2016， 硕士

【摘要】 青蒿素(Artemisinin)是从青蒿(Artemisia annua L.)中提取的一种具有独特过氧桥结构的倍半萜内酯。青蒿素联合疗法(ACTs)是世界卫生组织推荐治疗疟疾的首选方法。此外青蒿素衍生物对治疗病毒感染、癌症、免疫疾病也有重要的药用价值。然而,青蒿中青蒿素含量很低(约0.01%-0.8%),青蒿素分子结构较为复杂,很难采用全化学合成的方法生产青蒿素。因而克隆青蒿素前体合成途径上的基因,为青蒿素代谢工程提供候选基因和作用靶点就十分必要。MEP途径定位于植物细胞的质体中,为包括青蒿素在内的萜类合成提供基本前体物质异戊烯焦磷酸(IPP)和其同分异构体二甲基烯丙基焦磷酸(DMAPP)。2-C-甲基-D-赤藓醇-4-磷酸胱氨酰转移酶(MCT)、4-磷酸胞苷-2-甲基赤藓糖激酶(CMK)、2-甲基赤藓糖-2,4-环二磷酸合成酶(MCS)分别是MEP途径的第三、第四、第五个关键酶。MCT催化2-C-甲基-D-赤藓醇-4-磷酸(MEP)生成4-(5′-焦磷酸胞苷)-2-C-甲基-D-赤藓醇(CDP-ME);CMK催化CDP-ME合成4-(5′-二磷酸胞苷)-2-C-甲基-D-赤藓醇-2-磷酸(CDP-ME-2P);MCS催化CDP-ME-2P生成2-甲基赤藓糖-2,4-环二磷酸(ME-cPP)。本文首次克隆了青蒿MCT基因全长cDNA(AaMCT)、CMK基因全长cDNA(AaCMK)、MCS基因全长cDNA(AaMCS)并进行了相关生物信息学分析。AaMCT的cDNA全长1176 bp(GenBank登录号:KU365210),包含906 bp的开放阅读框,编码302个氨基酸。AaCMK的cDNA全长1462 bp(GenBank登录号:KT778821),包含1197 bp的开放阅读框,编码399个氨基酸。AaMCS的cDNA全长811 bp(GenBank登录号:KT725782),包含678 bp的开放阅读框,编码226个氨基酸。基因表达结果表明:AaMCT、AaCMK、AaMCS在青蒿分泌型腺体中大量表达,在叶、花、茎和根中少量表达;同时发现,AaMCT、AaCMK、AaMCS受到MeJA和SA诱导表达,MeJA处理后AaMCT、AaCMK、AaMCS基因的表达量呈极显著提高(P<0.01),SA处理后AaMCT和AaCMK基因的表达量呈显著提高(P<0.05)。亚细胞定位结果显示:AaMCT、AaCMK、AaMCS分别融合GFP都特异性定位在叶绿体中,这与MEP途径定位于质体这一事实吻合。最后,在拟南芥中分别超量表达AaMCT、AaCMK、AaMCS基因,拟南芥中叶绿素a、叶绿素b和类胡萝卜素含量都得到显著提高(P<0.01)。转AaMCT基因拟南芥三个株系的叶绿素a含量与对照相比分别提高了22.09%、16.60%、21.05%;转AaMCT基因拟南芥三个株系的叶绿素b含量与对照相比分别提高了23.70%、18.00%、17.43%;转AaMCT基因拟南芥三个株系的类胡萝卜素含量与对照相比分别提高了18.60%、11.53%、21.45%。转AaCMK基因拟南芥三个株系的叶绿素a含量与对照相比分别提高了23.83%、22.27%、18.26%;转AaCMK基因拟南芥三个株系的叶绿素b含量与对照相比分别提高了27.21%、22.79%、18.38%;转AaCMK基因拟南芥三个株系的类胡萝卜素含量与对照相比分别提高了25.24%、16.50%、15.53%。转AaMCS基因拟南芥三个株系的叶绿素a含量与对照相比分别提高了16.04%、12.69%、21.38%;转AaMCS基因拟南芥三个株系的叶绿素b含量与对照相比分别提高了16.18%、16.18%、20.59%;转AaMCS基因拟南芥三个株系的类胡萝卜素含量与对照相比分别提高了11.65%、10.68%、16.50%。表明AaMCT、AaCMK、AaMCS在萜类物质的生物合成中起重要作用。更多还原

【Abstract】 Artemisia annua L., a traditional Chinese medicinal herb of the Asteraceae well recognized for its synthesis of artemisinin. Artemisinin was the key ingredient of front-line antimalarial drugs. Artemisinin-based combination therapies(ACTs) are the most useful treatment for the cerebral and chloroquine-resistant malaria recommended by the World Health Organization. Moreover, the semi-synthetic derivatives of artemisinin have great medical value in virus, cancer and autoimmune disease. However, the content of artemisinin in the plants is very low and chemical synthesis is difficult. Therefore, it is meaningful to clone the gene on artemisinin precursor biosynthetic pathway and provide candidate genes for artemisinin metabolic engineering.The plastidial methylerythritol phosphate(MEP) pathway provides 5-carbon precursors, isopentenyl diphosphate(IPP), and its allyl isomer dimethylallyl diphosphate(DMAPP), for the biosynthesis of isoprenoid(including Artemisinin). 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase(MCT), 4-(cytidine 5’-diphospho)-2-C-methylery-thritol kinase(CMK) and 2-C-methylerythritol-2,4-cyclodiphosphate synthase(MCS) is the third, the fourth and the fifth enzyme of the MEP pathway, respectively. MCT catalyzes 2-C-methyl-D-erythritol-4-phosphate(MEP) to form 4-(Cytidine 5′-diphospho)-2-C-methyl-D-erythritol(CDP-ME). CMK catalyzes CDP-ME to form 4-( cytidine-5′-diphospho)-2-C-methyl-D-erythritol-2-phosphate(CDP-ME-2P). MCS catalyzes CDP-ME-2P to form 2-C-methylerythritol-2,4-cyclodiphosphate(ME-c PP).The full-length MCT, CMK, MCS c DNA sequence(Aa MCT, Aa CMK, Aa MCS) were cloned and characterized for the first time from Artemisia annua L. The full-length c DNA of Aa MCT(Gen Bank accession No. KU365210) was 1176 bp containing a 906 bp open reading frame(ORF) encoding a polypeptide of 302 amino acids. The full-length c DNA of Aa CMK(Gen Bank accession No. KT778821) was 1462 bp containing a 1197 bp open reading frame(ORF) encoding a polypeptide of 399 amino acids. The full-length c DNA of Aa MCS(Gen Bank accession No. KT725782) was 811 bp containing a 678 bp open reading frame(ORF) encoding a polypeptide of 226 amino acids. Tissue expression pattern analysis revealed that Aa MCT, Aa CMK, Aa MCS was highly expressed in glandular secretory trichome and poorly expressed in leaf, flower, root and stem. Aa MCT, Aa CMK, Aa MCS was found to be a methyl jasmonate(Me JA)-induced genes, the expression of Aa MCT, Aa CMK, Aa MCS was significantly increased after Me JA treatment(P<0.01). The expression of Aa MCT and Aa CMK was significantly increased after SA treatment(P<0.05). Subcellular localization indicated that the GFP protein fused respectively with Aa MCT, Aa CMK, Aa MCS was targeted specifically in chloroplasts. The Arabidopsis thaliana respectively overexpressed with Aa MCT, Aa CMK, Aa MCS showed significantly increase in the content of chlorophyll a, chlorophyll b and carotenoids(P<0.01). In the three transgenic lines with Aa MCT overexpression, the content of chlorophyll a respectively increased 22.09%, 16.60%, 21.05%, the content of chlorophyll b respectively increased 23.70%, 18.00%, 17.43% and the content of carotenoids respectively increased 18.60%, 11.53%, 21.45% compared with control group. In the three transgenic lines with Aa CMK overexpression, the content of chlorophyll a respectively increased 23.83%, 22.27%, 18.26%, the content of chlorophyll b respectively increased 27.21%, 22.79%, 18.38% and the content of carotenoids respectively increased 25.24%, 16.50%, 15.53% compared with control group. In the three transgenic lines with Aa MCS overexpression, the content of chlorophyll a respectively increased 16.04%, 12.69%, 21.38%, the content of chlorophyll b respectively increased 16.18%, 16.18%, 20.59% and the content of carotenoids respectively increased 11.65%, 10.68%, 16.50% compared with control group. In all, it can be concluded that Aa MCT, Aa CMK and Aa MCS plays an influential step in isoprenoid biosynthesis.更多还原