| Mangroves are an assortment of tropical and subtropical trees and shrubs, which have adapted to the inhospitable zone between sea and land. Mangroves form an unique mechanism of salt tolerance as they grow in high salinity environment. Proteomic analysis of its salt tolerance is of great significance and is a new mode for us to understand the salt tolerance mechanism of mangroves.Avicennia marina and Bruguiera gymnorrhiza were used in our experiments. These mangroves contain various secondary metabolites and a large amount of salt, and these substance have bad effects on the protein extraction and the isoelectric focucing (IEF). Thus, it is necessary to develop an efficient protein extraction method and two-dimensional electrophoresis (2-DE) condition before performing the proteomic analysis.In this study, we compared five different methods, i.e. trichloroacetic acid (TCA)/acetone powder, TCA/acetone precipitation, acetone precipitation, phenol extraction (Phe), and phosphate buffer extraction to determine their efficiency in extracting total proteins from A. marina leaves by 2-DE analysis. Using Phe method, high-quality and large-number of proteins could be extracted and satisfactory 2-DE maps with high reproduction, resolution power of protein and more clear background were obtained. It suggested Phe method could remove salt from the protein effectively and was practicable for mangrove protein extraction. Ampholine was used in IEF in this experiment, and the optimized condition for mangrove proteins 2-DE was:just using one Ampholine(pH5~pH8) and the total Vh was 14 kVh (for silve staining) or 16 kVh (for commassia brilliant blue). Under this condition, the protein plots in 2-DE maps were abundant, dispersed and clear, suggesting that the 2-DE technique developed in this study could absolutely meet the requirement of further proteomics study.Total proteins were extracted from A. marina and B. gymnorrhiza leaves 1 d and 60 d after NaCl treatment. We had used 2-DE and matri-assisted laser desorption/ ionization-time of flight-mass spectrometry (MALDI-TOF/MS) to identify proteins that are differentially expressed in response of NaCl treatment. From 2-DE data, 35 resolved differentially expressed proteins were detected in A. marina and 33 were detected in B. gymnorrhiza. Selected spots were subjected to tryptic digestion followed by identification using MALDI-TOF/MS. We determined 31 and 28 peptide mass fingerprinting (PMF) respectively in A. marina and B. gymnorrhiza. Searching in MASCOT database, 3 resolved differentially expressed proteins were identified in A. marina and 1 in B. gymnorrhiza. All of the identified proteins in A. marina are chloroplast proteins: ATP synthase beta subunit, phosphoenolpyruvate carboxylase(PEPCase) and maturase K. These proteins are related to increasing the salt tolerance by changing photosynthesis pathway or enhancing photosynthesis in A. marina and they are important for maintaining the function of chloroplast under NaCl treatment. The identified protein in B. gymnorrhiza was ADP-glucose pyrophosphorylase (AGPPase) small subunit. Its expression level was reduced by NaCl. The activity of AGPPase and the content of soluble sugars and starch were determined. The results showed that the activity of AGPPase and the content of starch were decreased by NaCl and the content of the soluble sugars was increased by NaCl. The reducing of starch producing and the accumulating of soluble sugars are propitious to increase osmotic adjustment under high salinity environment, that is one of the important way for mangroves to tolerate high salinity.This study optimized the protein extraction method of mangroves, and applied proteome method to study the proteins expression pattern of A. marina and B. gymnorrhiza under NaCl treatment. Some useful proteins were identified. This would contribute to the better understanding of the salt tolerance mechanisms in mangroves.
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