| Secondary metabolites were defined to opposed to the primary one firstly by Kossel in1891. Plant secondary metabolites play a vitally important role in the protection against biotic and abiotic stresses and are major sources for Pharmaceuticals, fine chemicals and food additives. Many of them have been investigated to have antibiotic, antiviral and antifungal activities, and hence can protect plants from pathogens. However, the contents of these compounds are generally extremely low (less than1%of dry weight). Chemical synthesis and plant cell culture have been developed to produce metabolites. But it is difficult to synthesize for their complex structural features and due to the internal competition of natural and unnatural precursors, yields of end-product is often low, concomitantly, it will raise difficulties in separation of these compounds. As for plant cell culture, the economy of a plant cell biotechnological production is the major bottleneck. In addition to the above method, many approaches based on the effect of stress were explored to boost the yield of the secondary metabolites in plants, the UV-B radiation is a kind of important elicitor.According to wavelength range, ultraviolet light was divided into three types: UV-A(320-400nm), UV-B (290-320nm) and UV-C (200-290nm). As the abiotic stress factor, it has effected the living beings on earth significantly. Due to the continuing depletion of the stratospheric ozone layer, the increasing UV-B radiation (280-320nm) on the earth’s surface caused an unoverlooked stress factor to plants, Acclimation to the changing UV-B environment can be expected to induce changes in the plant secondary metabolites, because a variety of these compounds in plants were capable of UV-B-absorbing. To date, approximately100,000secondary compounds were reported and identified from plants; among them, alkaloids is the second largest group. Alkaloids were found to posses appreciable absorption of the UV-B. However, the underlying molecular mechanism, of how the regulatory enzymes of different branches of secondary metabolic pathways to be coordinately altered by UV-B radiation, is still poorly understood. Proteome was defined as all kinds of proteins that a cell express in the particular physiological or pathological condition. Protein is the implementer of gene function, so the studies of protein structure, positioning and protein-protein interaction will provide direct basis for clarifying the essence of life. Differential proteomics is one important theme of the proteomics, containing the expression patterns and function patterns of specific proteins. It focused on filtering and identificating the difference between different species or different conditon in proteome. And it was one important tool to study plant physiology,development and genetic. So differential proteomics was a effective method to study the stimulation response and adaptability of medicinal plants, which will contribute to understand of the protein that the medicinal plant response to external stimuli and the related biosynthesis paths.Combinatorial hexapeptide ligand libraries(CPLL) is a new technique which reduces dynamic range of protein concentrations within a given sample.The device is commercially available as ProteoMine and has beenwidely used in several different fields, including human and animal proteomics. However, the application on plants is relatively few, except Arabidopsis thaliana, Pumpkin and Spinacia oleracea. The contents of enzymes involved in the secondary metabolism are generally low, which belong to the low-abundance proteins. In plants, the detection of low-abundance proteins are usually interferedby some high-abundance proteins, for instance, the Rubisco representing40%of total leaf proteins. Therefore, many methods were applied to enrich the low-abundance proteins, such as lectin columns, antibody affinity column, anti-phosphoamino acid antibodies and genetic engineering. The approaches of genetic engineering are mainly sequential, then step by step, purification, characterization and cloning of the enzyme, which are always expensive and time-consuming.CPLL is a cost-and time-effective tool that possessed much potential application value in plant proteomics, for example, the proteins that were not enriched by immunoprecipitation, might be useful with CPLL.Mahonia bealei (Fort.) Carr., a evergreen shrub from the genus Mahonia Nutt of the family Berberidaceae is one of important medicinal plants. Modern pharmacological researches indicated that it has many pharmacological activities including antimicrobial activity, anti-inflammatory, hypoglycemic activity, antioxidative activity and anticancer effects etc. Most chemical constituents of Mahonia bealei (Fort.) Carr. had been isolated, and the alkoids were regarded as main active components, particularly berberine, jateorhizine,columbamine and palmatine. However, traditionally the root of mahonia bealei(Fort) is collected and utilised as medicine, which is bound to severely impair the breeding and to cause the deficiency of plant resources, as well as to undermine ecological equilibrium.In the present study, a systematic study was conducted on the stress responses of Mahonia bealei (Fort.) Carr. to UV-B radiation. Alkaloid profiles were analyzed to determine the response to the certain band of UV-B and different incubation time of treatment. An overlay of UV-chromatograms from control and UV-B radiated plant leaves revealed substantial changes in quantity of several alkaloids. Compared with the control group, the maximum increasing of the content of jateorhizine, columbamine, palmatine and berberine were maximumly were919.6%,391.4%,192.4%and33.1%, respectively. The ordinary two-dimensional electrophoresis (2-DE), combinatorial hexapeptide ligand libraries (CPLLs) technology and RT-PCR technology was carried-out to address the underlying mechanisms that accounted for the alkaloids induction under treatment. By ordinary2-DE,13differential proteins were positively identified, but with CPLL,91low abundance proteins were detected. In the meantime, the six genes which encode the corresponding key enzymes in the alkaloids metabolic network of M. bealei were significantly up-regulated. |
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