The Function Study Of A HMG-box Transcription Factor,which Related With The Fruiting Body Formation Of Flammulina Velutipes

In this study,a transcription factor of HMG-box,which is associated with fruit body formation of Flammulina velutipes was identified based on genomic and transcriptomic data of flammulina velutipes.To investigate functional role of this transcription factor,a series of bioinformatics analysis were performed.On the basis of these analysis,overespression vector and RNA interference vector for HMG-box transcription factor were constructed.Through Agrobacterium Tumefaciens mediated transformation,overespression vector amd RNA interference vector of HMG-box transcription factor were transformed into Flammulina velutipes dicaryon strain 1123.Then we analysis the expression level and phenotype change of postive transformants.The main experimental results as follow:(1)Bioinformatics analysis of HMG-box transcription factor.The result of bioinformatics analysis suggests HMG-box transcription factor was located in the scaffold98,between position 207898-208877.The full length of HMG-box transcription factor sequence was 980bp(this transcption factor was hereafter referred to as fvhmgl).Fvhmgl includes 2 exons and 1 intron.No alternative splice form was found during the different developmental stages of flammulina velutipes.This transcription factor can be encoded 307 amino acid residues,predicted molecular weight was 34.157 kDa,isoelectric point was 5.39,and its mainly protein secondary structure was random coil.This HMG-box transcription factor contains a high mobility group box domain,its subcellular localization signal was located in the nucleus.The analysis of phylogenetic tree shows that this transcption factor has highly homologous with CC-XP-001836342 gene of Coprinus cinereus,and has some homologous with SC2660820 gene of Schizophyllum commune.The analysis of transcriptome data shows that the expression level of this transcption factor was up-regulated from the mycelium to the primordium period,after fruiting body formation its expression level was significantly down-regulated.(2)Construction of overexpression vector and RNA interference vector.Through enzyme digest-ligate,pBHg-BCA1-gpd was used as plasmid to construct overexpression vector fvhmgl-oe.Similarly,RNA interference vector fvhmg1-RNAi was successfully constructed using enzyme digest-ligate,with pBHg-BCA1-gpd as plasmid and PMD18-T as intermediate vector,respectively.(3)Transformation of Flammulina velutipes using Agrobacterium Tumefaciens mediated overexpression vector and RNA interference vector.4 positive transformants were obtained by transforming overexpression vector fvhmgl-oe and RNA interference vector fvhmgl-RNAi intoFlammulina velutipes dicaryon strain 1123 using Agrobacterium Tumefaciens mediated transformation.Of the 4 positive transformants,one overexpression transformant is named O1,while the other three RNA interference transformants are named R1,R2 and R3.(4)Expression level and phenotype change analysis.The gene expression level of four Flammulina velutipes positive transformants derived from real-time quantitative PCR shows that the expression level of fvhmgl in O1 was 90 folds as many as that of wild type strain 1123,while compared to wild type strain 1123,expression level of fvhmgl in R1,R2 and R3 was down regulated by 2.5,9 and 10 folds,respectively.Growth rate test shows that growth speed of hyphae of O1 was much slower than that of wild type strain 1123,while there was no significant difference in growth speeed of hyphae between R1,R2,R3 and wild type strain 1123.Cultivation experiment shows the hyphae of O1 was unable to form primordia,as well as fruiting body.Whereas the hyphae of R1,R2 and R3 was able to form fruiting body,besides,the stipe of their fruiting body were very stout,the cap of their fruiting body were very thick,and only had little lateral mushroom.The wild type strain 1123 can form fruiting body,but its stipe can not erect and short,its fruiting body was creeping like,and more lateral mushroom.Based on the abovementioned experiment result,we believe that fvhmgl plays an important role during hyphae growth of and formation of fruiting body.High level of expression of fvhmgl may supress growth of hyphae and formation of fruiting body,while low level expression is conducive to the formation of fruiting body.