Proteomic Analysis Of Salt Stress-responsive Proteins In Medicago Sativa L.

Soil salinity is one of the major abiotic constraints to agricultural productivity. It is reported that almost 5% of cultivated land is affected by soil salinity. Proteomic techniques are effective approaches that have been adopted to study the mechanism of plants response to salt stress and, to discover salt-responsive proteins. To investigate salt resistance mechanism for alfalfa, we used several techniques including two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).2-DE is one of the key techniques used in proteomic studies. In plants, protein extraction is a crucial step in the 2-DE analysis of proteins, and it can have a significant impact on both the quantity and the quality of protein detected. Using a modified TRIzol reagent method, we developed a simple and effective way to simultaneously extract DNA, RNA and proteins from a single sample. We verified that this method does not affect the quality or quantization of isolated DNA and RNA. Furthermore, we used 2-DE to compare M. truncate leaf, stem, and root samples processed using this new method with two commonly used methods. The results showed that our method was superior to the other methods, based on 2-DE patterns. This is the first report of a method developed for the simultaneous extraction of proteins and nucleic acids from the same plant tissue. The protocol can be used with plant sample preparation protocols for proteomic, transcriptomic, and genomic studies.Using the modified TRIzol reagent method developed, 7 days old Salt-tolerant alfalfa"Zhongmu.NO1"were treated with 0,100,200mM NaCl for 9 days. The proteome was separated by 2-DE and analysed by MS. Under Salt stress, 88 differentially expressed protein spots with higher than 1.5-fold spot density density were detected in gel image comparison using sivil staining, including 40 spots for leaf and stem , and 40 spots for roots. 70 protein spots were detectable by CBB staining and sampled for further MS analysis. The results showed that 53 proteins were successfully identified by MALDI-TOF/MS or MALDI-TOF-TOF/MS. These proteins were associated with a variety of functions, including photosynthesis, energy metabolism, metabolism, ROS, protein synthesis and degradation, cell growth/division, signal transduction, and transcriptional. Specifically, on the leaf and stem there were more different expression proteins were involved in photosynthesis and energy metabolism, while for roots were photosynthesis and energy metabolism.This is first report of proteomic study salt-responsive protein in alfalfa, and the result were do helpful for further stud the mechanism of plants response to salt stress.