The Analysis Of Differential Proteomics And Antioxidant Enzymes Activity Of Kenaf Leaves Under Salt Stress

Kenaf is a member of the Hibiscus in the Malvaceae family and native to east-central Africa, where it has been grown for several thousand years for food and fiber .Kenaf owns the characteristics of fast growing, high-yield, strong stress resistance and adaptability. To date, Kenaf fiber and by-product are used in the field of hemp spinning, paper-making, decorative material, earthwork cloth etc.This study mainly identified some relevant function proteins against salt stress of kenaf using proteomics method , and then preliminari understanding its protein function, exploring the mechanism of kenaf against salinity in the protein level. In addition, we analyzed the changing trend on antioxidant enzymes system activity of kenaf under salt stress condition and the coordination role between them, these all provide theoretical foundation for further researching the molecular and biochemical of kenaf in resistance salt stress and the utilization of saline-alkali, so as to guidance to the production cultivation of kenaf .Firstly, this study has establish a suitable and effective protocol of protein extraction for two-dimensional gel electrophoresis (2-DE) analysis in Kenaf leaf tissues by three extraction methods (Trichloroacetic acid/acetone method, Urea/thiourea method, Phenol extraction method) .Then the results were compared in regard to protein extraction efficiency, sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-DE gels. Furthermore, the 2-DE system had been optimized from 4 aspects: the pH range of IPG (immobilized pH gradient) stripes, sampling methods, sample volumes, and concentration of polyacrylamide gels. The data presented showed the phenol extraction method would be the best protocol to perform 2-DE analysis of Kenaf leaf protein, the protein extracted from phenol extraction method reached the purity of 26 protein bands in one dimension SDS-PAGE gels, in addition, 1374 protein spots on 2-DE gels were detected. The research also indicated that more clear background with a better distribution effect and a great many protein spots could be obtained on 2-DE gels under the conditions of active rehydration loading, 24cm IPG strips (linear pH gradient of 4-7), 1.4 mg sample, and 12% SDS-PAGE gels.Secondly, we measured changes in the proteome of kenaf seedlings that were exposed to salinity (70, 140, and 200 mM NaCl) using two-dimensional gel electrophoresis (2-DE) via liquid chromatography-tandem mass spectrometry. Approximately 1000 protein spots were visualized in the pH range of 4–7 through the CBB staining. Only protein identifications with score greater than P<0.05 were considered to be positive. Statistical analysis of the density of differential spots in the 2-DE gels. Experiment was performed in triplicate. To obtain detailed information about the proteome changes, we scanned the gels and digitized them using ImageMaster? 2D Platinum version 7.0. Fourty-two protein spots were significantly altered by exposure to the salinity environment. These proteins including Oxygen evolving enhancer protein, small heat shock protein, dehydroascorbate reductase, nitrate reductase and Superoxide dismutase and so on. Especially, S-adenosylmethionine-dependent methyltransferase was induced by salt stress, and six of the fourty-two proteins were up-regulation but the other thirty-six were down-regulation. These proteins were associated with a variety of functions, including energy and material metabolism, protein metabolism, photosynthesis, defense&ROS scavengers, transport and coenzyme metabolism. The down-regulation of Fructose bisphosphate aldolase expression by effecting the sugar and starch synthesis and accumulation may lead to growth inhibition of kenaf; the increased in 1,5 - bisphosphate carboxylase ribulose photosynthesis-related proteins would enhanced the photorespiration of Kenaf under stress to resist salt and the up-regulation of superoxide dismutase expression could increased the intracellular radicals. It is a common stress response that some differential proteins involved in metabolic processes , for helping the survival of kenaf under stress conditions, but also some metabolic processes may help to reduce the negative salt stress physiology. Under salt stress, kenaf can adjust the salt stress response protein by a variety of ways to affecting the metabolism, cell function, cell defense to maintain the relative ion balance and growth.Finally, we study the effect of the growth and the antioxidant enzyme activity of Kenaf seedlings under salt stress treatment , and then further to explore the physiological mechanism of salt tolerance. When kenaf seedlings were subjected to 0 mM and 140 mM NaCl for 3 d, 6 d and 9 d in half strength Hoagland nutrient solution and treated with different NaCl concentrations (0 mM,70 mM,140 mM,200 mM ) for 6 d, the fresh plant samples'weight, root length, stem length, leaf H2O2, malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and glutathione reductase (GR) activity were measured. The results showed that the effects of the salt stress of kenaf seedlings increased with time and salt concentration. SOD and POD activities increased slightly, or even decreased, while CAT and GR activities were relatively higher. CAT and GR may play the more important role in regulation of physiological and biochemical stress when responding to salt stress of kenaf seedlings. However, a variety of antioxidant enzymes in plant cells is not isolated, but interrelated, interacting with each other to form a complex antioxidant network to maintain the active oxygen generation and removal in a state of dynamic equilibrium.