| Most plant endophytes,especially actinomycetes,exhibit great potential in producing bioactive secondary metabolites.Given that few reports on the diversity and metabolites of endophytic actinomycetes in the Chinese medicinal plant Xanthium sibiricum,therefore,it is of great significance to carry out research on the endophytic actinomycete resources in Xanthium sibiricum.In the thesis,the high-throughput sequencing technology was firstly used for detecting the diversity of actinomycetes in different parts of Xanthium sibiricum;then,pure-cultural methods were used for exploring the diversity of culturable actinomycetes according to the diversity abundance of different parts.Meanwhile,the potential new species were identified using a polyphasic taxonomic approach;Finally,the antibacterial activity of the purified strains would be studied to pick out antagonistic strains of actinomycetes,and a preliminary separation and purification study was carried out on their fermentation products.The main results are as follows.(1)High-throughput sequencing results indicated that among the endophytes of Xanthium sibiricum,below the class level,the relative abundance of actinomycetes species(community composition analysis)was less than 0.5%,indicating that actinomycetes was non-dominant microorganisms;From the perspective of actinomycetes,the proportion of unidentified genus in the group Y(tender stem and leaf)and the group G(fruit)were 2.7% and 6.1%,respectively,indicating that the proportion of unidentified genus and species was large;In the group Y,there are 25 genus actinomycetes were detected without Kytococcus;16 actinomyces were detected in the group G without Actinoplanes,Amycolatopsis,Brachybacterium,Cellulomonas,Frigoribacterium,Gaiella,Ilumatobacter,Lapillicoccus,Streptomyces and Solirubrobacter.Nevertheless,all these results suggested that the diversity of endophytic actinomycetes in Xanthium sibiricum was very abundant.(2)In the present work,a total of more than 300 strains of actinomycetes were isolated and purified.In the basis of morphological observation and 16 S r RNA gene sequence analysis,they were merged into 58 different Streptomyces strains and one Nocardiopsis strain.According to cultural characteristics,58 Streptomyces strains were initially classified into 6 different color groups.A combination of cultural features,physiological and biochemical characteristics and phylogenetic analysis based on 16 S r RNA gene sequences,strains GGCR-6,CRPJ-33,CRPJ-51 and CR-Ben-6 could belong to potential new species(3)The taxonomic position of strain GGCR-6T was identified using a polyphasic taxonomic approach.All the genotypic and phenotypic data indicated that strain GGCR-6T represented a novel species of genus Streptomyces,for which the name Streptomyces aquilus sp.nov.was proposed.The type strain GGCR-6T= CICC 11055 T = JCM 33584 T.(4)Preliminary studies of antibacterial activity indicated that 24 of 58 Streptomyces strains had antagonistic effect to the selected target microorganisms such as Staphylococcus aureus,Escherichia coli,Aspergillus niger and Phyricularia grisea.Considering that strain CRPJ-33 could be a potential new species,it was selected for fermentation test.After fermentation,verification,concentration,Thin-Layer Chromatography and mass spectrometry analysis,30 different substances were obtained.Among of them,Arachidonic acid and genistein have direct and effective antibacterial or antitumor activity.It has been reported that clinically,genistein is mainly extracted from natural plants.As we all know,plant growth is affected by external conditions such as sunshine,etc.,and needs to occupy a lot of land resources.However,microorganisms,which grow and multiply quickly,are not restricted by external conditions,and do not need to occupy a large amount of land resources.Therefore,in this study,the discovery of genistein in the fermentation metabolites of strain CRPJ-33 is of great practical significance. |
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