Cloning And Functional Analysis Of Thmfs1, A MFS Efflux Pump Gene From Trichoderma Harzianum

Trichoderma spp are well known as biological control agents (BCA) activelyagainst a wide range of major destructive air-borne and soil-borne fungalphytopathogens. Major facilitator superfamily (MFS) transporters are secondarycarriers capable of transporting solutes in response to chemiosmotic ion gradients. Inthis paper we determained T. harzianum as material and we constructed the T.harzianum mycelium cDNA labrary. By screening the library, we firstly obtained anMFS transporter gene Thmfs1, which full-length of Thmfs1is2123bp, including a5’-terminal untranslated region of137bp, a3’-terminal untranslated region of294bpand an open reading frame of1692bp. The bioinformatics analysis data showed thatthe protein of Thmfs1contained14transmembranes and shared the same featurestructure domains with many well-studied MFS transporters in others fungi. In orderto further explore the function of Thmfs1, we constructed a Thmfs1disruption plasmidpBI121hΔThmfs1by using bianry vector pBI121h. Agrobacteriumtumeficiens-mediated transformation method was used to transform the disruptionplasmid into the T. harzianum to get the Thmfs1disruption mutant. We demonstratedby gas chromatography that the production of trichodermin in Thmfs1disruptiongroup (185.32mg/l) was decreased by less than17%compared to the parental strain,which meant Thmfs1wasn’t mainly responsible for trichodermin secretion. In vitroantibacterial experiment indicated that the fermanted broth from Thmfs1disruptionmutant had a less inhibited ability to the growth of five common pathogens and theirspore germination compared to the T. harzianum wild strain. We can draw theconclusion that gene Thmfs1discharges for a pump protein may be involved in T.harzianum secondary metabolites transportation, and playing an positve role in thebiocontrol activity. Tri5, coding a trichodermin synthase, was strongly suppressed bytrichodermin in a dose-dependence manner, while the expression of Thmfs1wasup-regulated obviously. Thmfs1disruption mutant displayed an raised sensibility toboth fungicides from the environment and itself, further study through in vitromycelial growth assay indicated that Thmfs1disruption mutant has a raised sensitivity to five chemical fungicide and self-secretion toxin compared to the parent strains. Wemay safely arrived at the conclusion that the efflux pump has a mutidrug resistence(MDR) function by discharging the toxins from the cell to the environment to protectthe body from the exogenous chemical fungicides and self-produciton toxins.