| There are three types of ultraviolet light according to different ranges of wave length which are UV-A between the wave length of 320nm and 400nm, UV-B between 290nm and 320nm, UV-C between 200nm and 290nm. As one of the various abiotic stress factors, UV light can affect and make changes in the physiological metablism and morphology of organisms on the earth through counteracting with DNA, proteins and various kinds of biological signal pathways. Vegetation is a very significant component on the earth which functions as maintaining the integrality of biological cycle and biosphere. For most plants, epicuticular wax layer, cuticles, pilus and other types of hard tissues always cover the surface of the plant body. Furthermore, plant tends to make adjustments through metabolism physiologically when encountered with adverse environmental stresses, so they are more adaptable than animals and microbes do when exposed on UV light. UV light has multiple impacts on plant body, for example, stunting growth, impairing photosynthesis and the synthesis of proteins. In order to resist the harmful effects of UV light, plants tend to produce a substantial amount of secondary metabolites. Those secondary metabolites including phenylpropanoids, alkaloids, terpenoids, etc., have big significance in preventing and treating human diseases and also are precious resources for discovering effective Traditonal Chinese Medicine drugs and natural medicines.Proteomics is defined as a study for all proteins exist in cells, tissues, organs and organisms or expressed by related genes, which covers the research of protein expression, peptide sequencing, protein structure and function, post-translational modification, protein-protein interactions, etc. It is one of important research fields in life science which is very closely related to genomics. Proteins play very significant roles in biological structures, growth, development, propagation and metabolism, and are the key reasons for bio-diversity. As the research of genomics is going forward, more and more understandings about it have been got. And researchers begin to notice that the study for proteins and enzymes which play direct functional roles in organisms seems to be very significant after the era of genomics..Clematis terniflora DC. belongs to the genus of Clematis, family of Ranunculaceae. Its aerial part is extensively used in the north part of Zhejiang province in China as a treatment for tumors and inflammatory symptoms in respiratory and urinary systems. Its main natural active compounds (secondary metabolites) are flavonoids and lignans.Our research focused on excised leaves of Clematis terniflora DC. which were exposed to UV light with different treatment time spans and different wave length ranges. A comparison of the HPLC fingerprints had been made between control and treatment to see effects of UV light on the secondary metabolites in this plant after which the prominently changed secondary metabolites were purified and with their structures elucidated. Through our research we found that, there were few changes of secondary metabolites after UV-A radiation. Whereas, after UV-B radiation for a period of time at the intensity of 120.83μw/cm-2, three new peaks appeared in the HPLC fingerprint compared with control. We used peak area as a preliminary index for the quantification of the three compounds to find that peak area of the three compounds tended to increase as the irradiation time span was prolonged. And we determined through more than three times of experiments that the best radiation time span was about 4 to 6 hours which gave the most remarkable changes. A substantial amount of leaves were irradiated by UV-B and then air dried. The research method of natural chemistry was used to separate and purify the three peaks and their structures were elucidated by UV, IR, NMR and MS which revealed three coumarins. They were 2H, 8H-Benzo [1,2-b:5,4-b'] dipyran-2-one,10-hydroxy-8,8-dimethyl-(9CI),4, 6,7-Trimethoxy-5-methylcoumarin and Luvangetin, respectively.In order to reveal the underlying mechanism of the UV effects on Clematis terniflora DC., we used 2-Dimensional Electrophoresis as a method for comparative proteomic study. Seventy-three differentially expressed proteins between treatment and control were obtained by Image Master 5.0 software analysis. Further identification of the seventy-three proteins were handled by MALDI-TOF-MS.In summary, the creative aspects of our study are described as follows:1) Trough the establishment of HPLC fingerprint, we had found that different wave bands of UV light had different effects on the secondary metabolites of Clematis terniflora DC.. And the prominently changed secondary metabolites were purified and structurally elucidated. Our results showed that a series of coumarins were induced by UV-B radiation in the leaves of Clematis terniflora DC., including both simple coumarins and pyranocoumarins.2) In the irradiation time span of eight hours, the effect of UV-B on Clematis terniflora DC.leaves increased as the irradiation time was prolonged.3) 2-Dimensional Electrophoresis was used for comparative proteomic study of the UV effects on this plant from which we had gained seventy-three differentially expressed proteins between treatment and control. MS identification of the seventy-three proteins led to the basic understanding of mechanisms underlying plant responses to UV-B radiation process.
|
PDF Full Text Request