Screening And Study Of Ansamycin And Halogenase Biosynthetic Gene In Valerian Endophytic And Rhizospheric Actinomycetes

The species,quantity and active metabolites of endophytic and root microbes in medicinal plants are relatively abundant,which is an important source of bioactive substances.The purpose of this study was to isolate endophytic and rhizospheric actinomycetes from Valerian and to screen the strains containing ansamycins and halogenase antibiotic biosynthesis gene clusters and to excavate new secondary metabolites.In this study,143 strains of endophytic and rhizospheric actinomycetes were isolated and purified from Valerian,and their antibacterial activities against bacteria and fungal pathogens were tested.The results showed that 22.4% of actinomycetes had a good inhibitory effect on pathogens.Secondly,based on the AHBA synthase gene and halogenase gene,the degenerate primers of ansamycins and halogenated antibiotics were designed to amplify the strains with good bacteriostasis effect by PCR.The results showed that strain AJ21 contains ansamycins antibiotic biosynthesis gene and strain AJ56 contains halogenase antibiotic biosynthesis genes respectively.Through 16 s rDNA sequence analysis,morphological observation,physiological and biochemical studies and phylogenetic analysis,it was found that both AJ21 and AJ56 belong to Streptomyces.Based on the antimicrobial activity result of AJ21 and AJ56 and antibiotic biosynthesis gene fragments,genomic analysis showed that: the genomic sequence of AJ21 was about 10 Mb,8415 coding genes and the GC content was 73.73%.The genomic sequence of the strain AJ56 was about 8 Mb,7259 coding genes and the GC content was 71.85%.By functional analysis of genes,it was found that both of them were resistant to tetracenomycin and had abundant secondary metabolic gene clusters,including 54 in strain AJ21 and 36 in strain AJ56.Combined with the above analysis,it was found that the strain AJ21 had good inhibitory effect and abundant secondary metabolic gene cluster.By optimizing the fermentation medium and fermentation days,a large amount of fermentation extraction was carried out.The results showed that the extract had a good bacteriostatic effect on Bacillus subtilis.Thesecondary metabolites of strain AJ21 contained actinomycin D by HPLC and mass spectrometry.The strain AJ56 contained abundant secondary metabolic gene clusters.The genetic transformation systems of AJ56 with S17-1/pSET152 and S17-1/pKC1139 were constructed under the conditions of final concentration of naphthyronic acid and Apr were respectively100 ?g/mL and 1.2 ?g/mL,which provides an experimental basis for further mining of its potential secondary metabolites.