Study On The Trichothecene Biosynthetic Genes In The Filamentous Fungus Trichoderma Brevicompactum

The filamentous fungus Trichoderma spp. exist widely in the air and the soil. The most frequently suggested Trichoderma mechanisms of biocontrol include mycoparasitism, competition, and antibiosis. In our previous work, an isolate strain0248was screened out from the plant endophytic fungi, and was morphologically and molecularly identified as T. brevicompactum. Our bioassay showed that T. brevicompactum0248was of high antifungal activity for phytopathogens such as Rhizoctonia solani and F. oxysporum. T. brevicompactum0248was capable of biosynthesizing the secondary metabolite trichodermin.In further study, it was found that trichodermin production was high in dextrose beef extract medium and no trichodermin was detected in a-lactose peptone medium by T. brevicompactum0248in each experiment. However, the biomass of T. brevicompactum0248has no significant difference in the above two cultivation media as the dry weights of T. brevicompactum mycelia were comparative after72h fermentation(2.73g·L-1in trichodermin-producing condition and2.67g·L-1in trichodermin-nonproducing condition).Since the biosynthesis mechanism of trichothecenes in Trichoderma is not clear, the transcriptome sequencing and gene expression analysis of T. brevicompactum0248under different culture conditions were carried out to obtain more information on T. brevicompactum metabolism for further increasing trichodermin yields.According to the transcriptome data, two unigenes, tri5and Unigene8037with potential roles in trichodermin synthesis and differential expression under different cultivation conditions of T. brevicompactum0248were chosen for validation using Real-time quantitative PCR (qRT-PCR).The results showed that a significantly different expression of tri5gene and U n i g e n e8037o c c u r r e d in t r ic ho d e r min p ro d uc ing a nd no n-p r o d u c ing me d ia. Th er e la t ive e xp r e s s io n le ve l o f t r i5g e ne in t h e t ri c ho d e r min pr o d u c ing me d iu m w a s107.18t ime s h ig he r t ha n t ha t in no n-p ro d u c ing me d iu m a ft e r96ho u rs inc u ba t io n,a nd t he p r o d u ct io n o f t r ic ho de r min w a s e n ha nc e d w it h t he inc re a si n g e xp re ssi o nle ve l o f t r i5g e ne, w h ic h r e a c he d t o t he ma x im u m o f31.25m g· g-1. Th e re la t iv ee xp r e s s io n le ve l o f U n i g e n e8037i n t he tr i c ho d e r min pr o d uc ing me d iu m w a s8.73t ime s h ig he r t ha n t ha t in no n-p r o du c ing me d iu m a ft e r72ho ur s inc u ba t io n.T h er e s u lt s ind ic a t e d t ha t t r i5g e ne a nd U n i g e n e8037ma y ha v e a c lo se re la t io nsh ip w it h t r ic ho de r min me t a bo lis m in T. b r e v i c o m p a c t u m.T he tr i5ge n e w a s s il e nc e d us in g R N A i t e c h no lo g y in o rd e r to a na lyse it sf u nc t io ns. A d e t a ile d e le c t r o po r at io n me t ho d w as d e ve lo pe d to d e live r e ffic ie nt ly s iR N A int o T. br e v ic o m pa c tu m0248, b y c ho o s ing d if fe re nt c o nd it io n s fo r t hevo lt a g e. T he s po r e s o f T. b r e v ic o m p ac tu m w a s e le c t ro tr a ns fo r ma t e d b y d sR N A-tr i5,t he ph e no t yp ic c h a nge s in t r a ns fo r ma nt s w e re o bs e rv e d, t he re la t ive e xpr e ss i o n o ftr i5ge ne w a s a na ly z e d b y q R T-P C R a nd t he tr i c h o d e rm in y ie ld w a s d e t e ct e d b y G Cme t ho d. A ft e r e le c t r o tr a ns fo r ma t e d b y d s R N A-t r i5, t he g ro wt h o f t ra nsf o r ma t e ds t r a in w e r e inh ib it e d w h ile t he ne g a t ive c o ntr o ls w e re no r ma l. Th e re la t iv ee xp r e s s io n le ve l o f t r i5g e ne in t ra ns fo r ma t e d s tr a in t re at e d u nd e r800V w a s0.75t ime lo w e r t ha n t ha t o f t he ne g a t ive c o nt ro ls a ft e r96ho u rs inc u ba t io n. Th e re su lt sd e mo n s t r at e d t ha t tr i5g e ne is e s s e nt ia l in t ri c ho t he c e n e s bi o s ynt he si s.