| Chloroplast that is considered as the power plant for life activities in plant,is one of the most important organelles for plant cells.Then,it will have far-reaching significance to study the changes of physiological status and biochemical activities of chloroplasts in adverse conditions.In our preliminary works,we have found that the enhanced UV-B stress caused dysfunction in wheat seedlings.While,He-Ne laser irradiation could protect seedlings against UV-B stress.In this study,we used winter wheat(Triticum aestivum L.)seedlings as the research object,through indoor simulation enhanced UV B radiation environmental conditions,analyzed the effects of the UV-B radiation enhancement(10.08KJ/cm2)and He-Ne laser(?=632.8nm,5.23mW/cm2min)irradiation on seedling chloroplast physiological status and chloroplast proteomics,in order to further clarify the protective effects and cellular mechanism of the low dose of He Ne laser treatment on UV-B radiation damage in wheat seedlings.These results will have important theoretical and practical significances to further improve protective effect of He-Ne laser.Main results are as follows.(1)Our experiments found that the enhanced UV-B radiation commonly caused wheat seedling chloroplast dysplasia,resulting in the significant changes in morphology and ultra-structure and chemical composition of chloroplast.UV-B stress also reduced photosynthesis efficiency,altered chlorophyll fluorescence parameters.While low doses of He-Ne laser irradiation effectively alleviate the UV-B radiation induced damage effect on wheat seedling chloroplasts.(2)Enhanced UV-B radiation significantly caused ROS accumulation of the wheat leaves,which was one of the important reasons resulting in cell toxicity and cell death.The formation of these ROS products mainly resealed through chloroplasts received UV-B stress.He-Ne laser irradiation can reduce the release of chloroplast ROS,then reduce the toxic effects of the UV-B stress on wheat cells.(3)Wheat seedlings separation of the complete chloroplast and chloroplast protein two-dimensional electrophoresis systems have been established via the exploration and optimization method.A complete and high purity of chloroplasts can be obtained in 30.0%,45.0%,60.0% of the discontinuous sucrose density gradient centrifugation.After TCA-acetone extraction,two-dimensional electrophoresis of protein is conducted,by the use of 17.0cm,inter-public linear strip pH4~7,the sample amount is 280.0?g/gel,13.0% SDS-page gel concentration,the results showed that the separation of chloroplast proteins are better.Wheat chloroplast proteins were separated via solid-phase IEF/SDS-page two-dimensional gel electrophoresis technique.The two-dimensional electrophoresis figures have obtained by dyeing stain and color silver stain methods,respectively.(4)400 or so clearly protein spot were identified via PD Quest software analysis,on the basis of the analysis results of 30 significantly protein expression differences point the MALDI-TOF peptide mass fingerprint analysis,and combining the software and Uniprot KB/Swiss-Mascot prot database search,25 different proteins were identified,which primarily involved in the chloroplasts of photosynthetic carbon metabolism,energy metabolism and antioxidant defense,and other important physiological processes.In addition,there are two proteins were identified as Uncharacterized protein,and we speculated that the molecular functions associated with UV response and protective through database retrieval.(5)Six protein spots which are closely related to chloroplast photosynthesis,energy metabolism and antioxidant defence physiology were selected to Q-PCR experiments and are compared with those of the protein expression level analysis.We found that a few protein points cut trend in transcription level rise,with its protein expression level of almost the same change trend,further verify the experimental results of proteomics.These proteins are involved in the regulation of diverse hormones,protein transport,protein interactions,the accumulation of reactive oxygen species and leaf growth in vivo for wheat seedlings,so we speculated that the protective and repair effects of He-Ne laser irradiation for enhanced UV-B radiation on plant cell injury may be related with the regulation process of chloroplast protein expression models.(6)We selected one new key protein,namely the UV-B response protein gene(LOC100844138-UVB 2)as the research object,and successfully obtained 1752 bp genetic stripe through gene amplification and cloning.In the subsequent experiments,we will further test the sequences of the purposed fragment,and verify its biological functions by downstream engineering technology.All together,the enhanced UV-B radiation changed wheat seedling chloroplast protein expression spectrum,leading to abnormal occurrences of some important resistant protein expression,reactive oxygen species metabolism imbalance,which resulting in chloroplast structure and its biological function damage.However,low doses of He-Ne laser irradiation can protect wheat chloroplast from enhanced UV-B stress damage,the main reason of the laser biological effects is because of He-Ne laser activates a few of the UV resistant protein and UV-B damage repair protein expression,then,improves the wheat seedling chloroplast UV-B stress resistance and its repair ability. |
PDF Full Text Request