Screening Of Plant Endophytic Actinomycetes Producing Quinones And Research On Structural Determination And Biological Activity Of Furanonaphthoquinone

Quinones is a kind of important natural products, and can be found wildely in plants, bacteria, fungi, and other organisms. Recently, a wide range of biological activities, such as: anti-virus, anti-bacterium, antifungal and anti-cancer, anti-inflammation and swelling and the ability to kill parasites and so on werereported on anthraquinone by many scholars. For example,so far, major antitumor drugs in clinic use and under research are quinones. The concerntration ofeffectivecompound in plants is very low, coupled with complicated plant secondary metabolites causes serious ecological and environmental problem which hinder the research and applications of quinones.So far, many researchers reported that plant endophytes may have the same metabolism pathway and products with the host plant.In this work,11 actinomycetes enriching medium were used to isolate endophyte from the seeds of Trewia nudiflora. We successfully isolated 73 endophytic strains from the seeds of T. nudiflora. 66 isolated strains were confirmed to be actinomycetes by morphological and 16S rRNA sequence analysis based methods. Besides five strains have not been identified beause of failing of cloning of the 16SrRNA gene.Isolated strains were tested for antibacterial and antifungal activityusingStaphyloccocusaureus,Pseudomonasaeruginosa,Bacillussubtilis, Rhizoctoniasolani andGibberellas-aubinetii.Among the 66 strains, 40.9% (27 strains) strains can inhibite the growth of Staphyloccocusaureus, 53% (35 strains) have the inhibition effect on Pseudomonas aeruginosa, 60.6% (40 strains) have the inhibition effect on Bacillus subtilis, 42.4% (28 strains) have the inhibition effect on Rhizoctoniasolani, 18.2% (12 strains) have the inhibition effect on Gibberellasaubinetii. These result suggested that high perscentage of endophytic actinomycetes from the seeds of T. nudifloracan produce antibacterial and/or antifungal compound.Degenerate primer for polyketide synthase Type III (FNQ6) and carboxymuconate cycloisomerase (FNQ21) were used to sreen for endophytes containing key genes of biosynthesis of naphthoquinone. PCR result shows that 15 strain contain FNQ6 gene. But FNQ21 were only detected in S. sp. HTZ 27. Furanonaphthoquinone(FNQI) was identified from fermentation product of S. sp. HTZ 27 after solid fermentation, extraction, silica column fraction, HPLC purification, MS and NMR analysis. We got 5mg pure furanonaphthoquinone(FNQI) from 1 liter S. sp. HTZ 27 agar fermentation whichalmost equal to productivity ofS. cinnamonensis DSM 1042 reported Seeger Ket al.with optimized fermertation conditions.Experiment of antitumor activity of FNQI shows FNQI can preferentially inhibite the growth of tumor cells, i. e. A549, H460 etc., IC50for H460 is about4.5μg/mL similar with paclitaxe(lIC50for H460 is about5μg/mL after treating with 48h) in the same experimental condition and method of this work.This work has laidsolid foundation for study of the furanonaphthoquinone biosynthesis and regulation, the molecular mechanism of its bioactivities, i.e. anticancer, and commercial applications. At the same time, this work also got preliminary information of community structure of endophytic actinomycetes of Trewia nudiflora seeds, which provides valuable information to explore the origin and evolution of endophyte, interaction between endophyte and plant, the role of endophytein the biosynthesis of natural products of plant.