Salt Mustard Glutathione Peroxidase Stress Response Protein Expression Analysis

Glutathione peroxidases (GPXs) is one of the most important antioxidant enzymes in plants, which has great significance in clearing excessive accumulated active oxygen radicals and protecting membranes from oxidation. Studies of the whole GPXs in plants have developed from single gene cloning to GPXs gene family cloning, mainly focused on gene cloning, phylogenetic tree analysis and expression pattern analysis in RNA level at present. Our laboratory has focesed on ThGPXs. Gene sequences of eight members in ThGPXs have been achieved and their expression characteristics in responding to adversity stresses have been studied using qRT-PCR. Antioxidant enzymeses mainly play a role in protein level, thus, studying ThGPXs expression change in protein level has more important meaning to interpret functions of ThGPXs.Based on our previous researches, antibodies of ThGPXs were prepared according to ThGPXs amino acid sequence characteristics. ThGPX8was inserted into prokaryotic expression vector, and the conditions of the protein expression were studied. Two-dimensional electrophoresis (2-DE) combined with Western blotting was applied to research ThGPXs specific expressed characteristics. Constructing the plant expression vector with GFP fused ThGPXs lay the foundation for ThGPXs subcellular localization study. The results obtained as follows:1. Two ThGPXs peptide antibodies, TsGPX-A and TsGPX-B, were constructed based on amino acid sequence characteristics of ThGPXs. Both of the antibodies has been detected by SDS-PAGE and showed good specificity.2. ThGPX8was inserted into pET-28a (+) prokaryotic expression vector which contains His-tag, the best induced expression conditions of ThGPX8were explored, and Western blotting was utilized to analyze ThGPX8expression under different conditions. The results showed that construction of pET-ThGPX8was successful, which induced by IPTG expressed a protein about23kD, and that induced at37℃for5h transformed E.coli could obtain higher expression quantity. The research laid a foundation for ThGPX8purification, protein expression and function.3. Total proteins extracted from leaves was separated by2-DE. ThGPXs were detected by use of Western blotting. Comparing the effect of alkaline phosphatase (AP) with enhanced luminescence (ECL) in detecting ThGPXs, experiment system of2-DE combined with Western blotting to detect ThGPXs expression was preliminary set up.4. Treated with20%PEG6000for6h,24h and48h, respectively, total protein of Thellungiella leaves were extracted respectively, untreated materials was as control. Total protein was tested by2-DE and Western blotting in order to study the expression characteristics of ThGPXs under the stress of drought. Based on the predicting ThGPXs molecular weight range,15protein points of expression volume difference more than2.0times were received;5.By inserting ThGPXs(ThGPX1、ThGPX2、ThGPX3、ThGPX6、ThGPX7) into plant expression vector containing reporter gene GFP, the pRTL-ThGPXs were constructed successfully, and it would lay a foundation for subcellular location of ThGPXs.In this study, ThGPX8prokaryotic expression vector was constructed. Meanwhile,2-DE and Western blotting technology were explored to research and identify the expression of specific proteins, aiming to set up the experiment system for further research on ThGPXs response to stresses in protein expression characteristics.