| In response to the scientific and technical issues involved in the scientific concept of "new origin,new technology,new strains,new genes,new products,new uses",the colorful Gypsum Forest in Yuanjiang,the earth Forest in Yuanmou and the colorful Sand Forest in Luliang,were selected as research sample regions.Combined with cultivation-dependent and cultivation-independent methods,the research plan revealed the diversity of actinobacteria in three regions and their correlation with environmental factors and established regional actionbacterial resource bank and species catalogue through antibacterial activity and screening of various types of functional genes.Furthermore,genomic sequencing of valuable representative strains was carried out and the synthesis of genetic clusters of secondary metabolites was predicted,in order to provide a new valuable strain of resources for the development of leading compound drugs.The research carried out has the scientific research value and the good application prospect,not only lays the theory,the method and the technical foundation for the development and utilization of the microbial natural medicine,but also provides the original strain kind for the use of actinomycetes resources.Firstly,soil samples were grouped according to sampling season,sample characteristics and sampling area,and then were analyzed by high-throughput sequencing analysis of 16S rRNA V3-V4,combined with environmental factors and soil microenvironment in different areas,to fully understand the composition and spatial distribution of bacteria,especially actinobacteria.Through species annotation,1,770,1,829 and 1,794 OTUs were obtained in the three sample areas respectively,which showed that all the three samples had abundant species diversity and richness,especially actinobacteria belonged to dominant bacteria in the three samples regions,and more than 80 actinobacteial genera were detected.Under the influence of environmental factors,the species abundance of actionobacteria in the three sampling regions was quite different.Rubrobacter and Arthrobacter were the highest in Yuanjiang area.In addition to Arthrobacter,Intrasporangiaceae and Pseudonomicaceae also occupied a large proportion in Yuanmou soil samples.Micrococcales and Solirubrobales were the dominant actinobacteria in Luliang sand forest samples.The composition of actinobacteria in the three sample points showed obvious differences in time as well as in space.The results of high throughput sequencing result showed great potential for the development of actinobacteia resources and provide valuable information for pure culture and isolation.Secondly,actinobacteria were isolated and identified from soil samples of three sampling region by selective isolation method.A total of 1381 strains of actinobacteria were obtained,including 394 in Yuanjiang sample area,545 in Yuanmou sample area and 442 in Luliang sample area.These actinobacteria are distributed in 11 orders,24 families and 60 genera,reflecting rich species diversity.Each sample also shows its unique community structure of culturable actinobacteria.In addition to the large difference in the proportion of common actinobacteria,the specific genus were isolated.On the basis of abundant community diversity,many potential new taxa were obtained in each sample area:29 in Yuanjiang,44 in Yuanmou and 37 in Luliang.Five of them were classified and identified by polyphasic taxonomy method.Three new species of Kineococcus were identified:Kineococcus gypseus sp.nov.Kineococcus terrestris and Kineococcus aureoles sp.nov.A new species of Prauserella:Prauserella flavalba sp.nov.and a new species of Glycomyces:Glycomyces terrestris sp.nov.Finally,more than 600 strains of actionobacteria were screened for antimicrobial activity(Gram-positive/negative bacteria,fungi)and functional genes(NPRs/DTS/ABHA/CHX/LNM/SAL/Rpf/PKSE).The results showed that actinobacteria isolated from three special regions in Yunnan probided with great antimicrobial activity potential and genetic diversity of secondary metabolites.The resistance or genetic differences of the genus not only provide a large number of strain resources for the development of natural products,but also the exploration of its metabolic mechanism will help to understand the ecological and evolutionary knowledge of microorganisms.The gene secondary gene metabolite gene clusters of 7 strains were predicted by antiSMASH.The results showed the abundance and diversity of secondary metabolite gene clusters of different actinobactial species the most prominent of which was YIM 122051 strain.There are 59 biosynthetic gene clusters,and many gene clusters have high similarity or identical core genes with known natural product gene clusters with research value,and have good research value and development potential.In addition,based on the information of biosynthetic gene clusters,new compounds were isolated from strain YIM 121209-2.To sum up,the three selected region in this study contain abundant actinobacterial resources,unique geographical environment,geomorphological characteristics and environmental factors with large differences,which provide abundant strain resources and gene pools for the development of natural products,and are worthy of in-depth research and development. |
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