The Differential Proteomics And Gene Expression Analysis Of Response To Low Temperature And Weak Light Stress In Radish(Raphanus Sativus L.)

Vegetables are the absolutely necessary food of life, and Radish (Raphanus sativus L.) is a vital root vegetable crop of Brassicaceae which originated in China. Because of the large cultivated areas, high nutrientive values, it plays a key role in the vegetable production supply of China. Low temperature is a key fact that affects the crops yields and quality, as the energy source of photosynthesis, solar light is the basis of the plants survival, and the photosynthesis of plants depends on the light.Low temperature and weak light (WL and LT) stress, as a major abiotic stress, affects the growth and development, geological distribution and the yields of crops severely. Proteomics plays an increasingly important role in the post-genomics times, and as a supporting technology of proteomics,2-DE is,applied in many fields of biological research. Currently, there are few researches on the mechanisms of radish exposed to WL and LT stress. In this study, we use the ’NAU-JLQX’ leaves exposed to different hours of LT and WL stress as sample, perform the2-DE to study the differential expression on the proteomic level and use MS analysis to analyse differentially expressed protein spots; DDRT-PCR was performed to separate the differentially expressed bands of radish exposed to WL and LT stress; semi-quantitative-RT-PCR was exploited to analyse the LT and WL responsive genes on the clone and transcription level; molecular clone technology was used to clone low temperature responsive genes. And the main research results were as follows:The ’NAU-JLQX’radish leaves were used as samples and2-DE was performed to compare2leaf proteins extraction methods:Tris-HCl/TCA-Acetone method and PEG Fractionation method. And the results indicated that with the modified PEG Fractionation method, the number of protein spots of three fractions (F1, F2and F3) separated by2-DE were approximately537,170and852, while the protein spots number of TCA-Acetone method is only640. So, with the PEG Fractionation, pH4-7linear IPG strips, and1000μg/350μL sample volume, the effect of2-DE profile was the optimum.In order to investigate how Radish adapts to low temperature(LT) and low light stress(WL), a proteomic approach based on two-dimensional electrophoresis was adopted to identify proteins that changed in abudance in2-true-leaf-stage radish leaves during exposure to LT(5℃/2℃) and WL(100μmol m-2s-1)(treated for0h,24h and4days). A total of8up-regulated protein spots and11down-regulated protein spots were identified by MALDI-TOF mass spectrometry analysis. The identified proteins mainly participate in carbon metabolism (oxygen-evolving enhancer, RuBisCO large subunits, RuBisCO small subunits, RuBisCO ssu precursor, carbonic anhydrase), signaling (NDPK2), protein synthesis (60S acidic ribosomal protein), ascorbate recycling (dehydroascorbate reductase), and plant defense (cysteine protease). Gene expression analysis of15different proteins by semi-quantitative RT-PCR illustrated that the mRNA level was correlated well with the protein level.Differential-displayreverse transcription-PCR (DDRT-PCR) was performed to separate the differential expression genes after LT (5℃/2℃) and WL (100μmol m-2s-1) stress treated for0h,24h and96h. A total of32TDFs were separated and17of them were highly homologous with some genes. And identified TDFs mainly participate in transcription, photosynthesis, defense and stress responsive genes, transcriptional effect, protein blinding, protein kinase and expressional regulation. All of the results indicated that as a complicated process, during the WL and LT stress, a large number of physiological and biochemical functions of radish were affected.Conserved domains of cold stress related genes of different crops were used to design specific primers, and the radish ’NAY-YH’ was used to perform PCR amplification. As a result, DNA and cDNA sequences of four cold stress related genes were named as RsCOR14,Rs-MYB4and RsLOS2. And the results of sq-RT-PCR indicated that, after LT and WL stress, the expression of RsCOR14, RsLOS2and RsMYB4genes increased, while the expression of RsMYB4reached at the peak point when treated for6hours.