细毛樟香叶醇合酶基因的发现和鉴定
其他题名Identification and Characterization of Geraniol Synthase from Cinnamomum tenuipilum
杨涛
学位类型博士
导师郝小江
2004
学位授予单位中国科学院昆明植物研究所
学位授予地点中国科学院昆明植物研究所
学位专业植物学
关键词化学型 细毛樟 香叶醇合酶 基因克隆 萜类合酶
摘要本论文以植物细毛樟(Cinnamomum tenuipilum Kostrem)精油的多样性为线索,选取三个化学型的细毛樟(芳樟醇细毛樟、香叶醇毛樟和金合欢醇细毛樟)作为研究对象,从叶精油的主成分是单菇(芳樟醉和香叶酉幻或倍半币欲金合欢醇)入手,利用分子生物学实验技术,从香叶醇叶片中找到并鉴定了香叶醇合酶的基因,此基因命名为CtGerS。根据已知的菇类合酶生物学信息,运用相似性克隆原理,从香叶醇化学型的叶片中得到一段420bP的cDNA片段,此片段进分析被归于裕类合酶家族。用3’RACE和5’RACE技术得到了全长的cDNA序列共2005 bp,开放读码框翻译603个氨基酸的蛋白,分子量为69.08kD,等电点为5.84。在基因数据库中没有检索到相同的序列数据。此基因有明显的N端质体定位信号肤序列,标识序列为双(X)8w,预示可能此基因是单萜合酶基因。此基因中有萜类合酶的底物结合和离子化序列DDXXD(aa337-aa341),而序列比较将此基因划分到Tpsb家族中,Tpsb家族的基因是被子植物单萜合酶。使用异源表达技术,将此基因以两种方式(全长和去除信号肽)构建到表达载体pET32a(+)中,用大肠杆菌BL21(DE3)g old菌株作为表达菌株,以融合蛋白的形式表达了相应的可溶性融合蛋白。经过单萜合酶、倍半萜合酶和异戊基转移酶的活性检测,利用GC/MS并且在排除了磷酸酶干扰后确定了去除信号肽的融合蛋白具有以GDP为底物合成香叶醇的功能。利用内标法定量,CtGerS以GDP为底物的ktn值为55 .8μM,酶反应依赖二价Mg2+或Mn2+离子。对CtGerS在三种化学型中进行表达模式的研究。RT-PCR结果表明CtGerS在三种化学型叶片中都有表达;NortAem blot结果显示CtGerS只在香叶醇化学型叶片中有明显的表达,而在芳樟醇化学型和金合欢醇化学型叶片中没有检测到信号。通过对CtGerS在三个化学型中的表达模式研究表明导致香叶醇主成分的CtGerS在香叶醇化学型叶片中有明显表达,而在其余两个化学型只有较低的表达量。此结果将化学型主成分的不同与形成主成分的基因直接联系起来。香叶醇合酶基因的发现和在三种化学型中的表达模式的不同提示细毛樟叶精油菇类主成分的不同可能是基因表达不同造成。 香叶醇属单褚,在植物中一般位于腺毛或分泌细胞中,细毛樟的叶片中有油细胞,而油细胞中富含各种挥发性油类物质,是细毛樟叶精油的主要储存地。CIGerS在细毛樟芳樟醇化学型叶片中的定位结果表明,CtGerS在油细胞中特异表达。此结果提示细毛樟其它叶精油中的菇类合酶表达部位也位于油细胞中,使以后对其它菇类主成分的研究提供一条思路。针对CtGerS的Southem blot分析表明OGelS有可能是以单拷贝的形式存在于细毛樟芳樟醇化学型的基因组中。综合本学位论文的实验结果和前人的研究得出结论:细毛樟香叶醇化学型的叶片油细胞中有大量的CtGerS的表达,导致香叶醇主成分的形成。CtGerS(注册号AJ457070)CtGers部分基因组序列(注册号AJ586886)
其他摘要To studies the reasons of essential oil diversity in Cinnamomum tenuipilum, three chemotypes (Linalool chemotype, LCT; Geraniol chemotype, GCT; Farnesol chemotype, FCT) were used as experimental materials. With the characteristic that linalool and geraniol belong to monoterpene and farnesol belongs to sesquiterpene, a novel gene was cloned and charactered as a geraniol synthase from the leaves of GCT. The gene was named CtGerS (C tenuipilum geraniol synthase). A similarity-based cloning strategy yielded a 420bp cDNA clone with the bioinformation of terpene synthases. This approach, by overlapping 3'RACE and 5'RACE with confirming full-length amplification, yielded a single cDNA of 2005 bp which encodes a deduced protein of 603 amino acids (aa) with a mass of 69.08 kDa and calculated pi of 5.84. The cDNA appeared to encode a typical N-terminal plastidial targeting sequence (RRXsW), which is anticipated for a monoterpene synthase, and the expected DDXXD element in the C-terminal domain (aa337-aa341), which plays an essential role in substrate binding and ionization. The result of sequence alignment was phylogenetically grouped within angiosperm monoterpene synthases subfamily (terpene synthase b, Tpsb). pET32(a+) was chosen as the expression vector for use in E. coli BL21(DE3) gold cells. The full length ORF and truncated sequence were ligated with pET32(a+). Two recombinant proteins were detected the activity of monoterpene synthase, sesquiterpene synthase and prenyl transferases. After compared with the phosphatase activity the truncated recombinant protein showed the activity of geraniol synthase. Biochemical characterization of the partially purified recombinant protein revealed a strong dependency of Mg2+ or Mn2+, and an apparent Michaelis constant of 55.8 μM for GDP. The CtGerS expression pattern was applied in the three chemotypes (LCT, GCT and FCL). CtGerS was expressed in the leaves of three chemotype by RT-PCR. However the result of Northern blot showed that CtGerS was only detected in GCT leaves, in the others no signal was detected. The results of CtGerS expression pattern showed that the scalar of CtGerS was more in GCT than scalar of CtGerS LCT and GCT. The result accorded with the main compound in three chemotypes. The CtGerS was found and the expression pattern of CtGerS was different in three chemotype showed that the chemotype in C.tenuipilum may be formed by the expression of different terpene synthase. Geraniol is monoterpene and generally locates in gland cells or secrete cells in plants. There are oil cells that stores rich volatility materials in C. tenuipilum leaves. The results of in situ hybridization analyses of CtGerS in GCT leaves showed that CtGerS was expressed especially in oil cells. It is a clew that other terpene synthases also were expressed in oil cells in C. tenuipilum. On the basis of the results described in this thesis and reported previously, the following conclusion: The CtGerS was experessed in oil cells of GCT leaves and formed to geraniol chemotype C. tenuipilum. CtGerS (Accession No. AJ457070) CtGerS partial genomic gene (Accession No. AJ586886)
页数145
语种中文
文献类型学位论文
条目标识符http://ir.kib.ac.cn/handle/151853/700
专题昆明植物所硕博研究生毕业学位论文
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杨涛. 细毛樟香叶醇合酶基因的发现和鉴定[D]. 中国科学院昆明植物研究所. 中国科学院昆明植物研究所,2004.
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