| Sinorhizobium fredii RT19 is a fast-growing soybea rhizobium strain, which was isolated from saline-alkali region in Tianjin and can normally grow on TY medium containing 0.6 mol/ L NaCl. In this study, the profiles of proteins expesssion in different growth phases of RT19 have been obtained using the technique of proteome analysis with two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). An efficient ImageMaster 2D was used to reveal the number of protein spots corresponding from lag phase to stationary phase, varying from 398 to 516, which manifested kinetic state of proteome in cell. Primary strucure and identification of a home-keeping protein (A73) were obtained by MALDI-TOF/MS and databases. Protein A73 is a DNA repair protein, named RecN, which is indispensable protein in genetic recombination and recombination repair.The protein expression profiles of RT19 growing in exponential phase after being treated with no NaCl and lmol/L NaCl for 5 min and 50 min were analyzed by differentially displayed proteome. The results showed that the number of the resolved proteins were 481, 465 and 424 respectively, which were separated by 2-D PAGE, showing that the amount of the proteins were decreased with the extension of salt shock treatment. All together eighty-two proteins were differentially displayed in response to salt-shock. Among them, 26 proteins were induced while 23 ones were inhibited; 12 ones were up-regulated and 21 ones down-regulated. In addition, the appearance of differential displayed proteins responding to different salt shock periods was also demonstrated, illuminating that complex network of regulation exist in cell. Twenty-six induced proteins were analyzed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and identified by database searching. Among them, 20 ones were assigned and their functions were known, including salt stress proteins, or enzymes are related to salt stress, y-butyrobetaine and Iipopolysaccharide synthesis, metabolism pathway and signal transduction.In order to study the similarities and differences between long-term salt stress and salt shock of RT19, the differential proteomic approach was used to analyzed the protein expression profiles. The results showed that eighty-six proteins were differentially displayed in response to long-term salt stress, among them , 34 ones were inhibited and 22 ones down-regulated, and 5 ones up-regulated. About 25 salt-induced proteins were identified by MALDI-TOF/MS, and finally the function of 17 induced proteins were determined, including heat-shock proteins (GroES, Clp). ABC transporter, RNA Polymerase (P-subunit) and enzymes involved in signal transduction and metabolism. Meanwhile, 84 proteins were idifferentially expressed after salt shock 5 min and 50 min in late exponential phase. Among them, 29 proteins are up-regulated and 18 ones down-regulated, while 12 proteins are inhibited. 25 induced proteins were identified by means of MALDI-TOF/MS. The results showed that these proteins were closely related to heat-shock proteins (DnaK, Hsp60). ABC transporter, the elongation factors (EF-Tu) and the other enzymes involved electron transduction and metabolism. Finally, comparing long-termed salt stress with transient salt shock, there are 12 proteins induced in common,which means different stress regulatory mechanisms are inextricably linked to each other.According to the analysis and identification of differential displayed proteome, two key enzymes, ADP-L-glycerol-D- mannoheptose-6- epimerase and mannosyltransferase, were induced in different salt stress and salt shock for RT19, which commonly participate in the biosynthesis of L-glycerol-D-manno-heptose, a major component of lipopolysaccharide. By means of SDS-PAGE,the results showed that the constructure of lipopolysaccharide changed in different salt stress and salt shock. It is inferred that the structural change is required due to the adaptation of bacteria to environmental stress.
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