| The extremly high salt-alkaline environment in Lop nur is rich in a variety of halophilic actinomycetes,which is a great treasure for mining diversity of actinomycete species,biological activity,secondary metabolites and resistance mechanism of salinity.23 high saline soil samples were used to isolate,collect,and preserve actinomycetes and the obtained 262 halophilic strains were then subjected to 16 S r DNA clustering analysis,resulting in discovering 13 potential new species.Six of them were identified as new species of actinomycetes.Meanwhile,the screening of salt-tolerant characteristics,antimicrobial activity as well as the antibiotic biosynthetic genes,including PKS I,PKS II,NRPS and APH,was carried out.In addition,13 purified compounds were structurally elucidated from the fermentation broths of 6 strains of actinomycetes.Lastly,the response of an extremly halophilic strain of actinomycete to the high or low salty stress was explored by using transcriptome comparison analysis.The results in detail are as follows:1.The species diversity of halophilic actinomycetes from salt-alkaline soil collected from Lop nur and its surrounding areas.262 actinomycete strains were obtained from 23 soil samples,among which 5 of them were salt-tolerant and 257 strains halophilic,including 13 potentially new species or new taxons of actinomycetes with a similarity below 98.5%.By the 16 S r DNA clustering analysis,these actinomycetes were classified into 4 suborders,5 families,and 8 genera.The eight genera were Actinopolyspora,Glycomyces,Nocardiopsis,Saccharomonospora,Saccharopolyspora,Streptomonospora,Streptomyces and Amycolatopsis.Nocardiopsis was the major isolated actinomycete group from Lop nur salt lake,the yield rate was 37.04%,while Actinopolyspora was the majority in salt-alkaline soil in the surrounding area of Lop nur with a yield rate at 25.20%.2.Multiphase classification of new species of actinomycetes.Six new species of actinomycetes were identified by multiphase classification method.The strain of TRM 45123 was identified and named as Saccharopolyspora halotolerans.Strains of TRM 45387,TRM 45198 and TRM 45127 were new species of Glycomyces and named as Glycomyces tarimensis,Glycomyces xinjiangensis and Glycomyces lopnorensis,respectively.Strains of TRM 4668 and TRM 45658 were identified and named as Halopolyspora tarimensis and Halopolyspora lopnorensis,respectively.3.The screening of antimicrobial activities and biosynthetic genes of antibiotics.The screening results of biosynthetic genes of antibiotics from 262 actinomycetes indicated that the positive rates of PKS I(polyketide synthases ?),PKS II(Polyketide Synthases ?),NRPS(Non-ribosomal Peptide Synthetase)and APH(Aminoglycoside Phosphotransferase)were 46.94%,25.57%,22.90% and 21.76%,respectively.PKS I and PKS II genes were existed in each genus while the probability of occurrence of PKS II was significantly less than PKS I.Most of actinomycetes carrying PKS I genes did not have PKS II gene.NRPS genes were mainly distributed in genus of Actinopolyspora and Streptomyces.75 strains of actinomycetes showed antimicrobial activities against at least one pathogenic bacteria with a positive rate of 28.6%.These active strains were distributed in Streptomyces,Nocardiopsis,Actinopolyspora,Saccharopolyspora,Glycomyces,Streptomonospora,Saccharomonospora with the positive rates at 14.5%,8.4%,7.3%,1.1%,1.1%,0.8% and 0.4%,respectively.Streptomyces was the best for finding out active strains.Each active strain contained one or more antibiotic synthetic genes.4.The isolation and identification of secondary metabolites from six actinomycetes.Thirteen compounds were purified by chromatograph methods and identified by NMR spectroscopic method from the fermentation broths of six actinomycetes.Their structures were elucidated as 3,4-dihydro-6,8-dihydroxy-3-methyl isocoumarin(45037-1),2-methyl-1,4-benzenediol(45037-2),bis(2-ethylhexyl)benzene-1,2-dicarboxylate(45037-3),1,6-dihydroxy-phenazine(45037-4),16?-methyl-11?,17?,21-trihydroxy-9?-fluoropregna-1,4-diene-3,20-dione-21-acetate(45306-1),?-Sitosterol 3-O-?-D-glucopyranoside(45306-2/45556-3),Nb-acetyl-tryptamine(45306-3/45380-1),N-(2-(1H-indol-3-yl)ethyl)-propionamide(45306-4),4,7-dihydroxy isoflavones(45556-1),4',5,7-trihydroxy isoflavones(45556-2/ 45214-1),soyasapogenol B(45214-2),1-heptyl-2-methyl-3-hydroxy-carbazole(45040-1),3-heptyl-4,5,6-trihydroxy-phthalide(45040-2).All of these compounds were firstly isolated from the secondary metabolites of these strains.5.A preliminary study on the response mechanism of a halophilic actinomycete to the salt stress.The experimental results of the transcriptome comparative analysis of the strain TRM 45611 under the condition of 8%(minimum salt concentration for the growth),17%(optimal salt concentration for the growth)and 25% Na Cl(maximum salt concentration for the growth)showed that the biological processes under salt stress include processes of cell metabolism,biological regulation and secondary metabolism.The cell components changed under salt stress were closely related to organelle and the transport metabolism of large molecular compounds such as proteins,nucleic acids,sugars or lipids.These metabolic processes were very important to the salt-tolerance of the train.The molecular functions responding to salt stress were closely related to the regulation of enzyme activity,molecule activity and transporter activity.Compared with 8% Na Cl as control group,78 genes were up-regulated and 155 genes were down-regulated with 17% Na Cl,while 383 genes were up-regulated and 302 genes were down-regulated with 25% Na Cl.In summary,the species diversity and main groups of actinomycetes in Lop nur region of Xinjiang were preliminarily revealed on the basis of the isolation and identification of the cultured actinomycetes.The antibiotic biosynthetic gene diversity,antibacterial activity and salt tolerance of the isolated strains were evaluated.The secondary metabolites of six strains were isolated,identified and resulted in thirteen structures.At the same time,the response mechanism of an extreme halophilic bacterium to the salt stress was studied at the transcriptome level.The above results pave a solid way for the protection and utilization of salt-tolerant actinomycetes in Xinjiang. |
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