Isolation Of Endophytic Actinomycetes From Several Anti-bacterial And Anti-inflammatory Plants And Study On Antibacterial Metabolic Potential

Actinomycetes have been an important source of new drugs and lead compounds because of its special habitats and have the ability to produce the same or similar compounds of host plants during the long evolutionary process.It has become a hot research due to its important role.In this study,fourteen kinds of medicinal plants with anti-bacterial and anti-inflammatory effect were collected from Chengdu,Kunming,Chongqing,Xiamen and Baoding as experimental material.Five kinds of media were used to isolate endophytic actinomycetes and 150 strains of endophytic actinomycetes were gotten and 50 endophytic actinomycetes were selected according to separation sources,colony morphology and cultural characteristics to make a comprehensive evaluation of its potential of antimicrobial metabolites by antimicrobial activity screening,and amplificating,sequencing and analysis of secondary metabolite synthetic gene(PKS-I,PKS-II and NRPS),and made phylogenetic analysis for active strains obtained to determine the species diversity of them.50 edophytic actinomycetes strains were fermentated using 5 different fermentation media(A,B,C,D,E)and fermentation broth was extracted with methanol,concentrated to dryness,dissolved in 1 m L 10% of DMSO and 250 kinds of fermentation extract were gotten.12 species of indicator bacteria and 3 species of indicator fungi were used to test antibacterial activity of 250 kinds of fermented extract.The results of the study showed that there were 48 endophytic actinomycetes haved inhibitory effect on at least one of the indicator bacteria or fungi,and 9 strains had antibacterial effect on more than six species of the indicator bacteria or fungi.Different fermentation media have an important influence on synthesis of active secondary metabolites.Secondary metabolite synthesis gene(PKS-I,PKS-II and NRPS)of 50 edophytic actinomycetes strains were amplified,sequenced and analysed.The results showed that four strains having PKS-I(8.0%)gene,twenty-four strains having PKS-II(48.0%)gene,four strains(8.0%)have NRPS gene,three strains(6.0%)have both PKS-I and PKS-II gene,three strains(6.0%)have both PKS-II and NRPS gene.Sequencing analysis showed secondary metabolite gene of some strains have lower similarity with the most similar sequences,for example,PKS-I gene of strain 190580 and strain 190600 had a closest similarity with the PKS gene(X86780)of Streptomyces hygroscopicus NRRL 5491,similarities were 79%;PKS-I gene of strain 181101 had a closest similarity with the PKS gene(DQ020253)of Streptomyces sp.UNSW 094300,similarities were 82%;PKS-I gene of strain EA36 had a closest similarity with the PKS gene of Streptomyces ambofaciens ATCC 23877 genome(CP012382),similarities were 86%;PKS-II gene of strain EA33 and strain TJ24 had a closest similarity with the PKS gene(KJ094408)of Micromonospora aurantiaca S97,similarities were 80% and 86%;PKS-II gene of strain EA34 had a closest similarity with the PKS gene(JX008012)of Nocardiopsis alba S78-1,similarities were 86%;PKS-II gene of strain 181101 had a closest similarity with the PKS gene(HQ698848)of Streptomyces sp.FXJ7.388,similarities were 84%;PKS-II gene of strain 190600 had a closest similarity with the PKS gene of Streptomyces venezuelae ATCC 15439 genome(CP013129),similarities were 88%;NRPS gene of strain TJ39 had a closest similarity with the NRPS gene(AY271636)of Amycolatopsis sp.AAL24,similarities were 75%;Suggested that PKS and NRPS genes of these strains had a novel genetic characteristics deserving further study.The 16 S rDNA sequences of 49 active strains having antibacterial activity or containing secondary metabolites synthetic gene were amplified and sequenced.The 16 S rDNA sequences were compared using Ez Biocloud and the phylogenetic relationship were analyzed,the results showed that 36 strains of 49 active strains belonged to Streptomyce,7 strains belonged to Nocardioides,5 strains belonged to Micromonospora,1 strains belonged to Glylomyces.This study showed that endophytic actinomycetes of medicinal plants with anti-bacterial and anti-inflammatory effect had good antibacterial activity and potential of secondary metabolite synthesis.This study showed that actinomycetes in anti-bacterial and anti-inflammatory medicinal plants had good antibacterial activity by antifungal activity testing and amplificating,sequencing and analysising of secondary metabolite synthesis genes,and different fermentation media had an important influence on the generation of reactive secondary metabolites.It laid a theoretical foundation for digging activity secondary metabolites by using a variety of media to diversify fermentation of endophytic actinomycetes in future.Some of secondary metabolite synthesis genes of tested strains had a novel genetic characteristics,which accumulated genetic resources for explore novel polyketides and non-ribosomal peptide compounds directionally.