Study On Secondary Metabolites Of One Strain Of Lichen-associated Bacteria

Lichens are classically described as an uniquely compound organism,which living in symbiotic association with fungi and alga or cyanobacteria.Lichens are able to survive in extreme environments,so they widely spread all over the earth.The discovery of Non-photoautotrophic bacteriobionts(Lichen bacteria)from lichens explored frequently according to the present study.The Alphaproteobacteria is predominant strain of lichen-associated bacteria that widely distributing in tissue clearance of lichens.Studies on the lichens present that they produce many of secondary metabolites with specific biological activities,which presented the lichenassociated bacteria is irreplaceable in the community of lichens.In this study,the bacteria strains with relevant bioactivities were selected on the basis of plate cultivation,and we confirmed the strain 8433 D that was isolated from Umbilicaria.esculenta.The 8433 D is the strain from the Rhizobiales bacteria clones of LAR1.This thesis is divided into two parts: firstly,the project adopted strategy of the Whole Genome Shotgun(WGS)to build libraries of different fragments.With the Next-Generation Sequencing based Illumina MiSeq,the de novo sequencing of 8433 D library were obtained using Paired-end sequencing.Then the sequencing data was filtered,sorted and evaluated of HQ contings in order to remove the sequences with joints.Using the anti SMASH and MiBiG to predict the metabolites diversity and biological activity through the analysis of gene clusters and the results showed that there is a gene cluster associated with biosyntheses of carotenoids metabolites while the key genes associated with multiple antibiotic synthesis.In the second part,we analyzed the physical and chemical properties of the secondary metabolites produced by lichen based on the product result of the first part.The bacteria were cultured using PDA medium in order to extract the products from ethyl acetate.Under the system of ethyl acetate-petroleum,the extract was fractionated to 7 components(Fr B-FrH)with column chromatography on silica gel.According to the results of TLC,we decided to isolate FrD,FrF and FrG by using ODS,Sephadex LH-20 and RP-HPLC,then 7 compounds(S1-S7)were received after purification.As a result,the structures of these compounds were inferred by mass spectrometry,nuclear magnetic resonance techniques and microspectral data,chemical studies indicate that the compounds include maackiain,tyroso and dipeptide compounds.The combination of de novo sequencing and extaraction technology of the secondary metabolites provides a new way to get extraction in lichen-associated bacterial.Compounds which have certain biological activity was obtained by this method.And combined with predictions of the analysis of gene cluster,we have enriched the research data about secondary metabolites of lichens.