| 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. |
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