Identification And Activity Evaluation Of The Secondary Metabolites Isolated From Tobacco Endophytic Fungus Paecilomyces Sp.

Plant endophytic fungi refer to a type of fungi that live in host plants and do not cause obvious disease symptoms to host plant tissues.Plant endophytic fungi have long-term symbiosis and interaction with host plants,leading to the same or similar synthesis pathways of secondary metabolites of plant endophytic fungi and host plants,which have become important microbial resources for the development of new active substances.Tobacco is rich in alkaloids,volatile oils and polyphenolic compounds,which have insecticidal,antibacterial,anti-inflammatory,anti-oxidant,and white blood cell-increasing activities,and tobacco endophytic fungi are rich in resources,but there are few studies on them at present.In this study,the tobacco endophytic Paecilomyces sp.TE-540 was used as the research object.The single colony separation method was used to isolate and culture the endophytic fungi,and the endophytic fungi were identified by combining morphology and molecular biology methods;then they were cultured,based on screening,we select a medium with richer metabolites for batch fermentation and culture to obtain fermentation extracts;separate the crude fermentation extracts to obtain monomer compounds;evaluate the activity of monomer compounds,and select compounds with better biological activity,and explore its mechanism of action.The specific test results are as follows:The strain was identified by morphology and molecular biology.The ribosomal r DNA gene transcription spacer sequence(ITS full-length sequence)of this strain was amplified and sequenced by PCR,and then compared with the existing strain information in the Genebank database through BLAST,it was found that TE-540 and the strain Paecilomyces sp.(Paecilomyces sp.,HG936908.1)similarity is99.9%.The strain was initially identified as Paecilomyces sp.The sequence has been submitted to the Genebank database and the sequence number is MT508829.Carry out medium-scale fermentation screening and batch fermentation.The molecular diversity of secondary metabolites of TE-540 under four different media(Sabouraud medium,PDB medium,malt extract medium,rice medium)was compared.Through HPLC-DAD spectrum analysis,it is found that the strains have more diverse compounds in the PDB medium.Therefore,it is determined to use PDB medium for fermentation.The separation and identification of compounds and the activity identification of some monomer compounds.A total of 27 monomer compounds(1-27)were isolated and identified from TE-540,including 1 rare adduct of mixed origin of sterol and polyketone(1),and 2 new compounds(16,23).Structure types include sterols,polyketones,pyrrole derivatives,diphenyl ethers,sesquiterpenes and xanthones.The activity test of monomer compounds 10 and 11 and the diphenyl ether compound Diorcinol N(DN,compound 22)showed that DN has significant anti-tumor activity.Researching on the mechanism of DN's anti-tumor activity and counting the inhibition rate of DN on six different cancer cells and normal,it is found that DN can significantly reduce the survival rate of A3 leukemia cells,and the sensitivity to normal cells is low.Therefore,we determined to explore the inhibitory mechanism of DN on A3 cells.Transcriptome analysis(RNA-Seq)found that a total of 9340 differentially expressed genes were found.Using gene cluster analysis,it was found that DN could up-regulate the expression of p21,p27,TGF-? and PI3K/Akt/m TOR,thereby causing autophagy.Counting the number of A3 cells in different cell cycles,it is found that DN blocks the normal progress of mitosis in the G1/S phase,thereby hindering the normal progress of cell division.In addition,using AO/EB staining,Hoechst 33,258 and monodansylcadaverine to label A3 cells,and observing through transmission electron microscopy,we found that DN caused cell vacuolation and autophagosome formation in a concentration-dependent manner.The above research results indicate that DN has the potential to become a drug lead compound for the treatment of acute lymphoblastic leukemia.In this paper,the structural diversity and anti-tumor activity of the secondary metabolites of tobacco endophytic fungi have been studied in depth,which provides a certain foundation and basis for the study of the medicinal activity of tobacco endophytic fungi,and also provides drug treatment for tumor diseases.A novel compound entity.