| With the deterioration of soil conditions, the studying tendency of the method to improving the soil salinization condition is increasing. Biological improvement, as an important means, attracts much attention. As well as the study of salt-tolerant microorganism is growing every year. Bacillus, as the strongest resistance microorganisms, was detected in the most ascendant salt-tolerant bacteria of many areas. So statistical analysis of the salt tolerance Bacillus can provide effective data reference for exploring the new salt tolerance strain. Separation and molecular analysis of salt tolerance strain can help us to understand the mechanism of salt tolerance, providing the germplasm resources for transgenic salt-tolerant crops, playing an important role in improvement and making full use of saline soil.In this paper, the temperature screening and gradient plate methods were used to isolate and salt-tolerant statistical analysis of culturable Bacillus strains from soil samples collected in various regions of the Northeast China.137Bacillus strains were obtained from500soil samples, in which74Bacillus strains showed4%-8%NaCl salt-tolerance,14showed8%-10%NaCl and2showed10%-14%NaCl.54%of total Bacillus strains showed more than4%NaCl salt-tolerant. The statistical results indicating that most of Bacillus had a high salt tolerance, the majority salt tolerance of Bacillus were less than8%NaCl.16S rDNA sequences of74Bacillus strains were amplified and analyzed for the determination of phylogenetic relationships.36salt-tolerant Bacillus strains were selected by sequences comparison and salt tests, which were different from each other in their16S rDNA sequences or the salt tolerance, showed99.7%-100%similarities with their closest type strains.17among them were submitted to GeneBank. The36strains classified into7species of Bacillus genera, Bacillus thuringiensis was majority (14strains,38.9%of all, highest salt tolerance were4-9%NaCl), second were Bacillus cereus (7,19.4%,4%-8%NaCl) and Bacillus subtilis (7,19.4%,8%-11%NaCl), then Bacillus anthracis (4,11.1%,5%-7%NaCl), Bacillus flexus (2,5.6%,9%-14%NaCl), Bacillus sphaericus (1,2.8%,5%NaCl) and Bacillus aryabhattai (1,2.8%,6%NaCl). Bacillus flexus and Bacillus subtilis were shown better salt tolerance. The analyses of LBR-4, the highest salt resistant strains (highest salt tolerance of14%NaCl), included morphological characteristics, physiological and biochemical characteristics, growth under salt stress and the phylogenetic of gene sequence. It was learned that this strain belongs to Bacillus, Bacillus flexus species, Gram positive halotolerant bacteria, and its growth temperature rang is4℃-40℃, the optimum is30℃; the growth pH range is7-10, the optimum is8; growth NaCl concentration range is0%-14%NaCl, the optimum is1%NaCl. The growth of this strain is affected by the high salt stress.In order to analyze the salt tolerance mechanism of LBR-4, we sequenced the genome of this strain and analyzed its results in this study. But didn’t get the similarly sequence to published salt tolerance gene fragments. Differences proteomics research of the whole expressed proteins of LBR-4were taken under the growth condition between normal and the highest salt concentration of14%NaCl. The results showed that the isoelectric points of most proteins of this strain distributed pH4-7, molecular weight distributed10-70kDa. The protein expression profiles of the strain had significant changes in the expression levels of118protein spots on the highest salt concentration condition of14%NaCl. Thereinto, the expression of78protein spots increased significantly, including22new proteins under the high salt stress,40expression levels with significant decreases,18disappeared proteins under high salt stress.6protein spots with significant changes in the expression levels were identified by mass spectrometry identification. According to the results, they are similar to Dihydroxy-acid dehydratase, Cell division protein FtsZ, FeS cluster assembly protein SufD, Uncharacterized carboxylase YngE, Putative aldehyde dehydrogenase DhaS and Phenylalanine--tRNA ligase alpha subunit respectively. According to MS result, analysis of the strain physiological changes that may occur under high salt stress... |
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