Proteomic Analysis Of Response To High Temperature Stress And Its Related Gene Identification In Radish(Raphanus Sativus L.)

Abiotic stresses including extreme temperatures, drought, flooding or chemical toxicity pose serious threats to agricultural production. However, High temperature, one of the most important abiotic stresses, is a destructive environmental factor that can reduce the yield and quality of crops. Radish (Raphanus sativus L.,2n=2x=18), a major annual or biennial root vegetable crop of Brassicaceae family, is one of the main vegetable crops in china. High temperature is the main limiting factor for radish production in summer. The important way for implementing year-round radish supply is breeding heat-resistant varieties. In recent years, the studies on physiological mechanisms under heat stress in radish have reported, but, have fewer reports on molecular mechanisms. In this study, compared proteomic approach was used to separate differentially expressed proteins in radish leaves and roots response to high temperature, identify novel genes by MS, analyze the expression changes in mRNA level of corresponding genes of differentially expressed proteins and clone novel heat-related genes; using mRNA differential display technology to isolate and identify the differentially expressed genes in radish under heat stress. Our studies will provide foundation for effectively improving stress tolerance and breeding heat-tolerant plant cultivar. The results were summarized as follows:Radish seedlings were put to a controlled growth chamber and subjected to a high temperature of40℃. After treated for0h,12h and24h, Tris-HCl/TCA-Acetone approach and phenol extracted approach were used to extract the total proteins of radish leaves and roots, respectively. The extracted samples were separated by2-DE, and visualized by CBB staining. Furthermore, the Mass Spectrometry (MS) was used to identify and analyze the differentially expressed protein spots. Totally,12protein spots were successfully identified which were highly reproducible in repeated experiments. Of these differentially expressed proteins, five were heat shock proteins (HSPs)(CPN10, HSP22, HSP17.4, HSP17.611and putHs42), four were related to energy and metabolism (FRK, THi1, LGL and OEE2), two were related to redox homeostasis (L-APX) and one was related to signal transduction (ANN), respectively. The all corresponding genes of identified protein spots were further analyzed at mRNA level under differentially treated time (0h,1h,6h,12h and24h) by semi-quantitative analysis. Results showed that L-APX, FRK, THi1, LGL, ANN, HSP22, CPN10, HSP17.6II and putHs42presented the consistent expression patterns between their protein and mRNA levels after treatment for0h,12h and24h at40℃; however, HSP17.4and OEE2showed differential expression patterns between protein and mRNA level at least at one time point.The extracted total proteins from radish roots with phenol extracted approach were separated by2-DE, visualized by CBB staining and identified by MS. Totally,8differentially expressed protein spots were successfully identified which were highly reproducible in repeated experiments. Of these differentially expressed proteins, two were heat shock proteins (HSPs)(HSP70-2and HSP18.2), three were related to redox homeostasis (GST and PDI), one were related to metabolism (PGK) and one was related to expression regulation (MRT04) and one were related to protein synthesis (Ras), respectively. Totally7(HSP70-2, MRT04, PGK, GST, RAS, HSP18.2and PDI) corresponding genes of differentially expression proteins were further analyzed at mRNA level under differentially treated time (0h,1h,6h,12h,18h and24h) by semi-quantitative analysis. Results showed that HSP70-2, MRT04, PGK, GST, RAS, HSP18.2and PDI presented the consistent expression patterns between their protein and mRNA levels after treatment for0h,12h and24h at40℃and differential proteins represent differentially expressed manners.Differential-display reverse transcription-PCR (DDRT-PCR) was applied to isolate heat-responsive related genes under high temperature stress with differentially treated time. A total of26TDFs were successfully cloned and22TDFs showed high sequence similarity to the genes of known or putative function, such as photosynthesis and respiration related genes, metabolism-related genes, stress-responsive related genes, protein-binding related genes and expression regulators related genes. Result showed that plants rapidly and simultaneously change the expression of a series of differential biological processes in order to response to heat stress, suggesting that a multitude of physiological processes involve in thermal stress response.In this study, the heat shock protein genes RsHSC70, RsHSP22and RsCPN10from radish were isolated using T-A clone. The full-length RsHSC70DNA sequence was2228bp, including two exons and one intron, contained a complete open reading frame (ORF) of1941bp, encoded for a protein of646amino acids with an apparent molecular weight of70.8kDa and an estimated theoretical isoelectric point of4.77. The full-length RsHSP22DNA sequence was865bp with no intron, contained a complete open reading frame (ORF) of594bp, encoded for a protein of197amino acids with an apparent molecular weight of22.4kDa and an estimated theoretical isoelectric point of4.93. The full-length RsCPN10DNA sequence was1172bp, including five exons and four introns, contained a complete open reading frame (ORF) of756bp, encoded for a protein of251amino acids with an apparent molecular weight of26.6kDa and an estimated theoretical isoelectric point of9.45. Semi-quantitative analysis was used to analyze the mRNA expression characteristics of the three HSPs genes under the same temperature with differentially treated time in radish leaves. Results showed that expression levels increased obviously after treated for1h at40℃and the three HSPs genes were induced by high temperature.