【作者】 杜娟；

【导师】 李彦舫；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 野生蹄叶橐吾[Ligularia fischeri(Ledeb.)Turcz.],嫩叶可食用,根及根茎入药名“山紫菀”,药理成分主要是萜类化合物紫菀酮、表木栓醇。该植物以其特有的营养价值和药用价值备受人们的关注。本研究以野生蹄叶橐吾为材料,通过引种栽培,进行蹄叶橐吾的组织培养,建立其遗传转化受体系统,首次尝试从功能基因和特异调控基因的角度深入研究紫菀酮合成的分子机制,克隆萜类化合物形成途径(甲羟戊酸途径)的重要调控点:HMG-CoA还原酶家族(HMGR、HMGR2),研究其基因特性、功能、表达部位;分析不同组织转录水平与紫菀酮含量相关性。成功构建了HMGR基因植物双元高效表达载体pBSHMGR。并利用农杆菌介导法对蹄叶橐吾进行了遗传转化。获得了转化效率极高的转基因蹄叶橐吾植株。PCR、Southern以及Northern检测结果表明HMGR基因已经整合到蹄叶橐吾核基因组DNA中,并在转录水平表达。转基因植株及其野生型对照蹄叶橐吾紫菀酮含量HPLC测定结果表明,HMGR基因的超表达增加了紫菀酮在转基因植株根、茎中的积累。为阐明蹄叶橐吾萜类化合物的生物合成代谢通路提供理论基础;为利用基因工程手段生产紫菀酮提供新的思路和理论依据。更多还原

【Abstract】 The genus Ligularia Cass is one of the large genera in Compositae- Senecioneae-Tussilagininae. In subtrib. Tussilaginac, Ligularia is closely related to, but more advanced than the genus Farfrrgizem Lindl. It includes six sections, 11 series and 129 species. All the taxa are distributed in Asia with only two species extending to Europe. There are 119 speciesin E. Asia, comprising 96 % of the world total. The triterpenoid glycosides are major chemical constituents of the genus Ligularia Cass., in addition that monoterpenes, sesquiterpenrs, diterpenes, steroids, coumarins, peptides are also isolated. Among of hem, some compounds possessed activities of antitumor, antibiotic, antiinflammation.3-hydroxy-3-methylglutaryl CoA reductase cDNA genes have been successfully isolated from Ligularia fischeri and named as HMGR with 2031 bp (GenBank accession number: DQ916106), which encodes a predicted polypeptide of 579 amino acids with 62.48kDa and pI 7.63 and HMGR2 with 1153 bp (GenBank accession number: EF133501), which encodes a predicted polypeptide of 343 amino acids with 36.33kDa and pI 8.44.The characteristics of HMGR and HMGR2 and their deducted amino acids were analyzed by the tools of bioinformatics in the following aspects: the composition of nuclei acid sequences and amino acid sequences, signal peptide, transmembrane topological structure, hydrophobicity or hydrophilicity, secondary structure of protein, molecular phylogenetic evolution and so on. The result s are as follows: the full-length gene of HMGR contains an opening readingframe, 5′and 3′-untranstrated regions; HMGR and HMGR2 are hydrophilic and transmembrane proteins which lack of signal peptide; the amino acid sequences of HMGRs include two functional HMG-CoA binding motifs and two functional NADPH binding motifs; the main motif of predicted secondary structure of HMGRs are alpha helix and random coil , beta turn and extended strand are spreaded in the whole secondary structure of protein.Southern blot analysis performed using a HMGR specific fragment as probe showed that two copies of HMGR gene in Ligularia fischeri. The RNA gel blot analysis showed a different HMGR expression patterns among the different organs at mRNA level with abundant amount in leaves. The determination of Shionone in roots and stems of Ligularia fischeri by HPLC showed rather high content of Shionone in leaves. To further investigate the function of HMGR, the overexpression of HMGR in Ligularia fischeri were alao studied by gene transformation via agrobacterium. PCR, Southern and Northern analyses of transgenic plants showed that the HMGR gene was integrated into the Ligularia fischeri genome and expressed at the transcriptional level. HPLC analysis showed that the Shionone content in transgenic roots and stems of Ligularia fischeri were increased 16.67% and 12.25% as compared with the control. It was suggested that overexpression of the HMGR gene could increase the content of Shionone and indicated that HMGR may play an important role in biosynthesis of Shionone.The cloning and characterizations of the above two genes would offer the theoretical basis to further investigate metabolic pathway of isoprenoid biosynthesis in plants.更多还原