Primary Study Of Differential Proteomics And Physiological Characteristics Effects Of Nitrogen Stress On Cotton Seedlings Root

The fertilizer application is very important for cotton growth, especially nitrogen fertilizer. The reasonable application of nitrogen fertilizer is the research hotspot in recent years and improvement of the utilization rate of nitrogen fertilizer has been becoming the current main research targets. Plant roots act as plants’ "source" and "sink" and are more attractive area in the ecosystem and global change research. But the nitrogen fertilization between root growth and physiological response of the complex and regulatory mechanism is not clear. Proteomics research is a proper and a direct way to help understand the nature of life activity. Therefore, research of the difference of cotton root protein level under nitrogen stress, combined with mass spectrometry and bioinformatics knowledge, obtain the information and function of differential proteins, explore the inner relationship between these proteins and nitrogen stress to be of great significance in understanding cotton nitrogen stress response mechanism. At the same time, it can provide a theoretical basis for improving the utilization rate of nitrogen fertilizer. This study selected lu 28 Bt-cotton as the experimental material, was carried out under two hydroponic experiments. First trial set up four N levels, including the N0 : 0 mmol/L, N2: 2 mmol/L, N4: 4mmol/L and N8: 8 mmol/L. The second test removed the N0 treatment, and research cotton seedling root morphology, physiological characteristics and differences of protein under N2, N4, N8. The main results are as follows:1. The morphology and dry matter of cotton root under different nitrogen levels were determination and scanned. The results showed that the morphological traits in N2 such as total root length, root surface area, and root volume were significantly lower than N4 and N8, and the morphological indexes of N8 processing reached the highest in each period. The accumulation of dry matter of N4 and N8 were more than that of N2, the root-shoot ratio of N8 showed a trend of increase after the first reduction, the root system and aboveground growth of N4 is coordinated, the root-shoot ratio of N2 increased. The low nitrogen inhibits the growth of root system, and high nitrogen promoted the root growth.2. Root activity reflects the life activities and absorption capacities of root. With the increase of nitrogen concentration, the root activity and nitrate reductase content were increased. Sufficient nitrogen supply can keep root activity and the content of nitrate reductase in a higher level.3. Screening total protein extraction method of cotton root including ammonium acetate saturated phenol-methanol method, TCA/acetone method and improved TCA/acetone/SDS method were concluded. The purity of protein sample obtained by ammonium acetate saturated phenol-methanol method is high, the protein spots is the most, reached 601, and the background of the image is clear, So this method is most suitable for extracting total protein of cotton seedling root.4. To test the different protein sample amount, the extractied protein samples obtained by the method of ammonium acetate saturated phenol-methanol were applied for two-dimensional electrophoresis. When electing 24 cm, pH4-7 interpublic strip, the sample amount is 1500μg, got 2-DE images of high quality. By analyzing the map through using Imagemaster software, the sample amount for 1500μg getting 720 points is more 160 points than the sample amount for 100μg. Selecting the saturation of phenol-methanol ammonium acetate to extract root total protein and the sample amount is 1500μg, then applied for two-dimensional electrophoresis can obtain ideal gel maps.5. Using the modified 2-DE method to separate the total proteins of roots under different nitrogen levels, the differentially obvious protein points were identified based on LC-MS/MS analysis. Together there were 17 protein points successfully to be identified, and there were six protein points to be upregulated expression while 11 to be downregulated expression under the nitrogen stress condition. Function analysis showed that they were mainly involved in the carbohydrate metabolism, photosynthesis, cell defense, amino acidsand protein synthesis, cells processing, molecular partner and other processes.