【作者】 郭惠明；

【导师】 林敏； 程红梅；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2009， 博士

【摘要】 紫茎泽兰(Eupatorium adenophorum. Sprengel)是一种入侵性很强的恶性杂草,起源于墨西哥和哥斯达黎加一带,它具有生命力强、传播速度快,植株本身毒性大,对土壤养分的吸收性强,分泌化感物质等特点,目前已经广泛分布于世界热带、亚热带30多个国家和地区,对森林、农田等生态系统造成的严重威胁,对入侵地区的经济、生态和社会造成了重大损失。该恶性杂草于20世纪40年代由中缅边境传入我国,在国家环保局首批定为最重要的16种外来入侵物种中,紫茎泽兰名列首位。目前国内外对于紫茎泽兰的研究主要集中在其入侵模式、生态学特性、防除方法和利用等方面。紫茎泽兰化感物质在生物入侵过程中发挥了重要的作用,其作用机制可能是通过影响植物光合作用、各种代谢以及根系生长和对矿质元素吸收等许多方面来实现入侵。然而目前,对于与次生代谢相关的化感物质的代谢调控机理方面的研究还很少,本文就是围绕这一点开展研究工作。针对紫茎泽兰基因的克隆和功能分析建立了研究平台,通过开展紫茎泽兰的化感相关的代谢途径的研究,为这种恶性杂草的综合治理提供理论依据,以期能够找到调控化感物质代谢的关键酶的编码基因,发掘一些能够为农业生产所利用的新基因。本论文的研究结果如下:1.构建了紫茎泽兰叶片cDNA文库。通过SMART的方法合作构建了高质量的紫茎泽兰cDNA文库。原始文库的滴度为1.23×107 cfu/mL,重组率达98 %,插入片段平均大小在900 bp左右,扩增文库滴度达到了1×1012,覆盖率达99 %以上,利用该文库筛选得到了紫茎泽兰核酮糖二磷酸羧化酶基因(RuBP)的cDNA片段(GenBank ID:FJ752421),表明文库质量合格。2.建立了紫茎泽兰的遗传转化体系。通过农杆菌介导的方法建立了紫茎泽兰的遗传转化体系。经过多种检测手段的鉴定,目前已经获得7株转基因植株,转化效率达到2 %。3.化感相关基因的克隆、筛选及表达谱研究。克隆了4个紫茎泽兰的基因片段,分别是3-羟基-3-甲基戊二酰辅酶A合成酶(HMGS)基因(GenBank ID:FJ913887)、1-去氧木糖-5-磷酸异构酶(DXR)基因(GenBank ID:FJ752422)、倍半萜烯环化酶(TS)基因(GenBank ID:FJ747651)和查尔酮合酶(CHS)基因。荧光定量PCR的结果表明,萜类代谢途径中的两个关键酶基因(HMGS和DXR)的表达水平,没有因为羟基泽兰酮的诱导而表现出显著的差异;CHS基因的表达水平,除了在羟基泽兰酮处理浓度为1.0 mM时的差异不显著之外,在其他处理浓度均有不同程度的下调,说明该基因与化感物质(羟基泽兰酮)的代谢途径相关。4. EaCHS1基因全长的克隆及表达载体构建。通过RACE技术,首次克隆了紫茎泽兰查尔酮合酶基因的全长(GenBank ID:FJ913888),将其命名为EaCHS1。EaCHS1全长1178 bp,其中5’非翻译区27 bp,3’非翻译区200 bp,最大开放阅读框架948 bp,编码316个氨基酸,理论分子量为33.9 kDa。根据EaCHS1基因的3’非翻译区序列设计引物,进行Real-Time PCR检测,结果发现:经过不同浓度羟基泽兰酮处理,EaCHS1基因的表达水平出现不同程度上调,只在浓度为1.0 mM时的差异不显著。Southern杂交表明,EaCHS1基因在紫茎泽兰中不多于4个拷贝。针对EaCHS1基因构建了植物过量表达载体并利用紫茎泽兰CHS基因的保守序列构建了RNAi载体,分别转化紫茎泽兰和烟草;5.羟基泽兰酮对植物根系发育的影响。利用模式植物—拟南芥,初步探索了化感物质(羟基泽兰酮)对受体植物根系发育的影响。结果表明:拟南芥根系成熟区的根毛生长在羟基泽兰酮浓度为0.025 mM时,受到促进,而在0.1 mM时开始受到抑制;主根的生长受羟基泽兰酮的影响,存在一个明显的低浓度促进、高浓度抑制的现象。更多还原

【Abstract】 Eupatorium adenophorum is a kind of noxious invasive alien weed origin from Mexico and Costarica. It has some characteristics such as strong vitality and toxicity; it also can spread rapidly, absorb nutrients from soil strongly, and produce allelochemicals. At present, Eupatorium adenophorum has widely distributed about 30 countries and regions at tropical and subtropical zones. In these regions, it has threatened the ecological system of forest and farmland badly. As a result, it has caused serious loss in economy, ecology and society. This worst weed was firstly introduced into China from Burma in 1940s. In the 16 types of exotic invasive species confirmed by the Ministry of Environmental Protection of China, E. adenophorum is the first one as the most ecologically significant and potentially destructive species for China. Up to now, most of the study was focused on the invasion and dissemination patterns of E. adenophorum, its physiological ecology, prevention and utilization of the weed. Those allelochemicals of E. adenophorum play an important role in process of biological invasion. Its mechanism probably influences many aspects in plant, such as photosynthesis, various metabolisms, root growth and metal elements absorption. Little research was reported about regulation mechanism of allelochemical in the secondary metabolic pathways. Our research was carried out encircling this point.A research platform was constructed for gene cloning and functional analysis in Eupatorium adenophorum in this study to provide a theoretical basis for the all-sided control of this malignant weed through the research in metabolic pathway related to allelochemical. By the study, we try to find out the coding gene of key enzyme which is related with the metabolism of allelochemical and clone some novel genes which can be used in agricultural production. The results are as follows:1. A cDNA library was constructed by using leaves of E. adenophorum as tissue materials and through the switching mechanism at 5’end of RNA transcript (SMART) method. The titer of primary library is 1.23×107 cfu/ml, and 98 % clones are recombinants. The average size of insert fragments is about 900 bp. The titer of amplified library is 1×1012, and 99 % coverage rates are obtained. The RuBP gene of E. adenophorum was screened out from this cDNA library (GenBank ID: FJ752421), this mean that the cDNA library has the qualified quality.2. A effective protocol for the Agrobacterium tumefaciens-mediated genetic transformation of E. adenophorum with tender stem segments as tissue materials was constructed. Status of transgenic plants was confirmed by a variety of experiments including RT-PCR, PCR and GUS histochemical assays. The transformation efficiency in E. adenophorum was 2 %, and 7 transgenetic plants were obtained by now.3. Four genes related to allelochemical were cloned, screened and analysed. They are 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGS) (GenBank ID: FJ913887), 1-deoxyxylulose-5-phosphate reductoisomerase (DXR) (GenBank ID: FJ752422), sesquiterpene cyclase (TS) (GenBank ID: FJ747651) and Chalcone synthase (CHS). The real-time quantitative PCR results showed that the expression level of two genes (HMGS and DXR) did not be affected by HHO obviously, whereas, the expression level of CHS gene had down regulation of different degree, except in 1.0 mM of HHO’s concentration. It meaned that CHS gene is probably related with the metabolism of allelochemical (HHO).4. The Chalcone synthase gene (EaCHS1) of E. adenophorum was cloned by RACE method (GenBank ID: FJ913888). The complete nucleotide sequence of EaCHS1 gene contains 1178 base pairs, deducing 316 amino acids. It was comprised of 5’-noncoding region (27 bp), open reading frame (948 bp) and 3’-noncoding region (200 bp). The molecular mass of the deduced protein is 33.9 kDa. Using the sequence of 3’UTR in EaCHS1 gene as primers, the real-time quantitative PCR result showed that the expression level of EaCHS1 gene was up regulated when induced by HHO except for in 1.0 mM of HHO. Southern blotting shows that EaCHS1 gene has four copies in E. adenophorum. We also constructed the over-expression vector of EaCHS1 gene and RNA interference vector which used the conservative sequence of CHS gene in E. adenophorum as target sequence respectively, and then transferred them into E. adenophorum and tobacco.5. Arabidopsis Thaliana, as a kind of model plant, was used as receptor plant in order to preliminarily explore the root development affected by HHO. The result indicated that the growth of root hair was promoted in the root maturation zone of A. Thaliana when the concentration of HHO was 0.025 mM, whereas it was inhibited when the concentration of HHO was 0.1 mM and higher. This phenomenon showed that the growth of main root was promoted in a low concentration and inhibited in a high concentration of HHO.更多还原