药用植物金铁锁种质资源研究及其鲨烯合成酶cDNA的克隆
其他题名Study on the Germplasm Resources and Its cDNA of Squalene Synthase Coloning of the Medicinal Plant Psammosilene tunicoides
钱子刚
学位类型博士
导师吴征镒
2008-06-16
学位授予单位中国科学院昆明植物研究所
学位授予地点昆明植物研究所
学位专业植物学
关键词金铁锁 种质资源 鲨烯合成酶
摘要金铁锁Psammosilene tunicoides W.C.Wu et C.Y.Wu是石竹科金铁锁属的单种属植物。为主产于云南西北部、中部和东北部,同时分布于贵州、四川和西藏的国家二级保护植物,是“云南白药”的主要组成药材之一。为保证其药材内在质量稳定性、均一性,建立其资源描述规范,必须首先进行金铁锁的种质资源研究。 本文采用多学科的研究方法对特产我国西南部的濒危药用植物金铁锁从物种、居群、个体、细胞直至分子等各个生物学层次进行研究。描述了金铁锁的生物学特性并对其生态环境适应性和适生区进行分析,进行人工繁育和传粉特征研究、核型初步分析,揭示金铁锁遗传变异式样(遗传结构)以及初步分析其濒危机制,结合化学成分分离、分析方法对有效成分和标准提取物进行研究,并进一步对金铁锁有效成分产生的关键酶基因—鲨烯合成酶(PSS)cDNA进行了克隆。 通过形态观察发现金铁锁个体间的小苞片、花瓣等形态特征存在较大变异,而叶片长/宽比相对较为稳定。金铁锁对不同水分、营养物质环境的有较强的适应性。首次运用CLIMEX 1.1对全国222个点的气候和地理因素的研究分析表明,云南、贵州、四川、西藏大部地区为金铁锁的适生区。 传粉研究发现金铁锁为混合交配繁育系统,是虫媒异交植物,同时可以自花授粉,其繁殖方式为近交繁殖,传粉昆虫主要为中华蜜蜂、食蚜蝇等。而对金铁锁6个居群研究发现,染色体2n=24,基数为12;与已有报道金铁锁的染色体数目2n=28不同。金铁锁可以通过组织培养进行快速繁殖。 应用扩增限制性长度片断多态性(AFLP)方法对金铁锁种质资源的遗传多样性进行评价,表明金铁锁物种水平的遗传多样性丰富(PPB=86.00%,He=0.2831),但其居群水平的遗传多样性偏低(PPB=32.5%,He=0.0858);同时居群间存在显著的遗传分化:基因分化系数Gst=0.6977和Shannon’s多样性基因遗传分化系数Ist=0.6975。金铁锁的地理空间距离和居群间的遗传距离无明显相关性。而所得到的9条特征带在种内可以作为区别各个产地居群的特征指纹。促进了对金铁锁遗传多样性和DNA指纹特征的了解。 对金铁锁标准提取物的提取工艺进行研究。利用高效液相色谱技术,对影响提取工艺的主要因素进行分析。对其有效成分的进行初步研究,首次从金铁锁中分离得到丝石竹酸-3-O-(6/-O-甲基)-β-D-葡萄糖醛酸苷,选择其作为指标性成分,制备了标准对照品。建立了含量检测的HPLC方法,对不同居群金铁锁的丝石竹酸-3-O-(6/-O-甲基)-β-D-葡萄糖醛酸苷进行含量测定,表明居群间存在较大的差异。 同时,首次采用RT-PCR和RACE等方法分离得到的鲨烯合成酶全长cDNA,是金铁锁三萜皂苷生物合成途径中的关键酶基因,丰富了相关基因的研究内容。为次生代谢工程方面研究奠定了基础。在用金铁锁鲨烯合成酶氨基酸序列与其它各代表生物的序列聚类分析时,属于石竹科的金铁锁较独立于真双子叶植物类的其它种类而与单子叶植物纲植物的关系较近,表明其可能是在真双子叶类分化之前已分化出来的一个独立的分支。
其他摘要Psammosilene tunicoides W.C.Wu et C.Y.Wu is a monospecifc plant of the genera which mainly distributes in the northwest, middle, northeast Yunnan and also Sichuan, Guizhou, Tibet. It is now embodied in Red Book of Chinese Plant, and classified as second degree national priority protection plant. As a major component of ‘Yunnan Baiyao’, we must study on its germplasm resources for guaranteeing the high, stable quality of the medicinal plant and establishing the descriptors and data standard of its resources. Pasammosilene tunicoides W.C.Wu et C.Y.Wu is a endangered medicinal plant with distribute only in southwest China. Studies of morphology, cytogenetics, sequencing of AFLP, phytochemistry, biogeography, favorable eare, breeding, pollination were performed on the species, trying to reveal the divergence pattern and relationship among different geographical populations within the species and analyze its endangered reasons, more important to use isolating and analytic method researching the effective compounds and its standard extract. We have taken further steps to cloning the cDNA of a key gene relation to effective compounds----squalene synthase. There are discrepancy of the form of bractlet, petals etc. between individuals among population, but the shape of the leave is relatively stable by observing morphological characters. Psammosilene tunicoides has widely adaptation to moisture and nutrition. We have used CLIMEX 1.1 in first on the nation's 222 points of climatic and geographical factors of analysis, Tibet, Sichuan, Yunnan and Guizhou is favorable eare for it. Psammosilene tunicoides has a hybrid mating system. It is an allogamous, entomoplily and inbred reproduction plant, and can be self-pollination. The insect of pollination is Apis cerana, Eristalis tenax etc. According to the study on the plant of 6 populations, it has 24 stripes of chromosome, 2n=24, x=12, a quantitative difference from others` 2n=28. The plant can be propagated rapidly by tissue culture. Using the method of Amplified fragment length polymorphisms (AFLP) to investigate the genetic diversity of 9 populations of Psammosilene tunicoides, the result is that: genetic diversity of it is relatively high at the species level(PPB=86.00%,He=0.2831), but is lower within populations (PPB=32.5%,He=0.0858); There are extremely high levels of population genetic differentiation among populations: The genetic differentiation (Gst) is 0.6977 and genetic differentiation by Shannon’s diversity (Ist) is 0.6975. The distance among these geographical populations is not consequent on its genetic distance. We have found the 9 characteristic fingerprints that can be used to identify the geographical populations and supplied a deficiency of the research of its diversity and characteristic fingerprint. In order to establish the method for the quality control of the medicinal plant, the standard extract was prepared which set up with the experiment of multi-factors by HPLC technique. A primary phytochemical investigation led us in the first place isolate the gypsogenin 3-O-(6’-O-Methyl)-β-D-glucuronopyranoside, and choose it as characteristic constituent, prepare the standard compound, establish the method of determining effective content. We have gotten the result that there are difference of effective contents among the populations by the method of HPLC. For the first time a full-length cDNA fragment of Squalene synthase of Psammosilene tunicoides was isolated by the method of RT-PCR and RACE. Squalene synthase that can catalyzes the first enzymatic step in sterol and triterpenoid is a key regulatory point modifying the total triterpene saponins biosynthesis. This work provided an basis for the study on Psammosilene tunicoides Secondary Metabolism, and replenished the knowledge of the genes. Comparing the order of amino acid of Psammosilene tunicoides squalene synthase with the others`, we have gotten the result that Psammosilene tunicoides of Caryophyllaceae is close to monocots, and is a distant relative of eudicots, it may be occurred formerly as a distinct branch than eudicots.
页数183
语种中文
文献类型学位论文
条目标识符http://ir.kib.ac.cn/handle/151853/44
专题昆明植物所硕博研究生毕业学位论文
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钱子刚. 药用植物金铁锁种质资源研究及其鲨烯合成酶cDNA的克隆[D]. 昆明植物研究所. 中国科学院昆明植物研究所,2008.
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