Comparative Proteome Of Different Ecotypes Of Reed (Phragmites Communis Trin.) Adapted To Natural Droght And Saline Habitats

In the deaert regions of northwest China, Hexi Corridor of Gansu provice , Phragmites communis, a hydrophytic species whose typical habitats are the fresh and brackish water area of swamp and riversides, adapted itself to adverse terrestrial habitats and evolved into other three terrestrial reed ecotypes under natural selection pressure, and thus developed a unique habitat distributing four reed ecotypes(swamp reed, SR; light salt meadow reed, LSMR; high salt meadow reed, HSMR; dune reed,DR) within the scope of several kilometers. These stable reed ecotypes are ideal experimental system for mechanism research into plants tolerance or resistance to drought or salinity. In the present study, ISSR (inter-simple sequence repeat) and RAPD (random-amplified polymorphic DNA) markers were used to detect genetic diversity of 4 different ecotypes of reed so as to confirm the evolving trends originated from the physiological and ecological research. Secondly, in order to find the proteins involving in the reed ecotypes'tolerance and resistance to natural drought and salinity, proteomics and bioinformatics techniques were used to analysis leaf total proteins and chloroplast protein components of 4 different ecotypes of reed, to soil drought and salinity. The main results were summarized as follows:1. Nine effective primers were screened from 30 ISSR arbitrary primers, and a total of 99 DNA bands were amplified, among which 51 (51.5%) were polymorphic. Thirteen effective primers were screened from 45 RAPD 10-oligonucleotide arbitrary primers, and a total of 195 DNA bands were amplified, among which 87 (44.6%) were polymorphic. Genetic identity based on ISSR and RAPD data showed a positive correlation (r=0.845, P<0.05). Based on unweighted pair-group method with arithmetic averages (UPGMA) cluster analysis on DNA bands amplified, together with the correlation analysis between genetic distance and soil water contents and soluble salt contents as well, the present results suggest that the genetic diversity occurs among the four ecotypes of reed in adaptation to long term natural drought and salinity, showing an obvious evolutional tendency from swamp reed via salt meadow reed to dune reed.2. To optimize two-dimensional polyacrylamide gel electrophoresis ( 2-DE) system for the separation and quantification of plant protein species, a suitable 2-DE system was developed by selecting different components and concentrations in isolation solutions and lysis buffers, and by improving IEF and SDS-PAGE procedures originated by O'Farrell. This included sample preparation with water-saturated phenol and then mixed with 5 volumes of 0.1M ammonium acetate in methanol (NH4Ac/methyl).The isolated proteins were lysised in lysis buffer consisting of 8M urea, 2M Thiourea, 4% CHAPS, 2% Ampholine(pH3.5-10: pH5-8=1: 4)and 65mM DTT. Isoelectric focusing gels were made in glass tube (140×1.5mm inside diameter). 1.45mLGel mixture contained urea 0.825g,30% acrylamide 200uL,10% NP-40 300uL , ddw295uL, Ampholines 50uL ( pH3.5-10:pH5-8=1:4), 10% APS/TEMED1.6uL. The separated proteins in the 2D gel were detected by silver staining. Each protein sample (80μg) extracted from different plant materials, such as cultured rice, maize and the other 3 terrestrial ecotypes of reed growing in natural habitats, were separated and analyzed, and high-quality and quantity separation of leaf total proteins obtained by this 2-DE system. This 2-DE system is recommended as a suitable tool for analysis of plant proteomics.3. Two-dimensional gel electrophoresis was used to analysis protein expression profiles of leaf total proteins of 4 reed ecotypes. After electrophoresis,102 differentially expressed proteins were cut out, digested in gel by trypsin and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)..Based on the information of the mass fingerprinting (PMF) obtained from peptides for each protein spot, 25 of these 102 proteins were indentified by searching database online. This included fourteen Ribulose-1,5-bisphosphate carboxylase/oxygenase, six ATP synthase beta subunit , catalase and glyceraldehyde-3-phosphate dehydrogenase, which are much more abundant in three terrestrial reeds than swamp reed, showing an obvious evolutional tendency in adaptation to long term natural drought and salinity. Otherwise, some proteins, such as rubisco activase precursor and FtsH protease, were also identified. 4. Chloroplasts of swamp reed and dune reed isolated by Percoll gradients. Chloroplast ptoteins were analyzed by two-dimensional gel electrophoresis. 119 different protein spots were found, which were digested, analyzed and identified as described in leaf total proteins. Based on the information of PMF, 10 of these 119 discrepant proteins were identified by searching data from MSDB detabae onlin. Among the identified proteins, 3 spots are RuBisCO, 4 were subunits of ATPase. Moreover, V-H+-ATPase, ATPase family associated with various cellular activities (AAA-ATPase-like ) and many other lower matching proteins, such as cytochrome P450 monooxygenase and terpene synthase, were also identified. Interestingly, expressed abundance of the most identified proteins was higher in the dune reed ecotype than that in the swamp reed one, impling that these proteins might involve in dune reed's adaptation to drought habitat. And the result of RuBisCO large subunit and ATPaseβsubunit obtained from chloroplast proteins coincided with that of the leaf total proteins.