Preliminary Study On Structure And Function Of A Gene Vv-rcd1 In Volvariella Volvacea

Volvariella volvacea is cultivated on a large-scale in many tropical and subtropical districts and has highly edible and medical value.However,the cultivation of V.volvacea depends on seasons strongly,and it tends to open pileus and autolyze after harvest,which are obstacles to the production of V.volvacea.Moreover,research development on the formation and development of V.volvacea fruiting body goes slowly.Recent years,as the genome data released,the researches on its functional genomes gadually develop,which will propel the production and cultivation of V.volvacea.We obtained a fruiting body formation related gene deno minated as vv-rcdl,based on the genome data of V volvacea,transcripome data and related studies on Neurospora crassa.After the analysis of expression level of the gene,we established RNAi vector and overexpression vector,and obtained transformants through ATMT methods.We did preli minary study on the transformants of V.volvacea.The main results are reported as follows:1.The homologous gene of rcdl in V.volvacea was GME4331,deno minated as vv-rcdl.The full length of the gene was 1648 bp,the length of 3’UTG 12 bp,5’UTR 288 bp,coding region 1348 bp;the gene,containing 6 extrons and 5 introns,is a member of rcdl protein family,consisting of Armadillo-type fold domain.2.RCD1s from N.crassa,Schizosaccharomyces pombe,Oryza sativa,Mus musculus,Homo sapiens,Danio rerio,D.melanogaster,Arabidopsis thaliana,Caenorhabditis elegans and Saccharomyces cerevisiae shared high similarity with RCD1 from V.volvacea with a identify of 54.42%,when RCD1 from Neurospora crsssa shared a 61.28%identify with V volvacea.Moreover,RCD1 from V.volvacea had the same 5 basic sequences with the 10 model organism,which indicated that RCD1 has high conservatism in eucaryons.Phylogenetic analysis showed that rcdl spreads widely in edible and medical fungi,but that its function may vary largely in fungi with distant relationship.3.DGE tag mapping results of eight RNA specimens of the mycelia,primordia,stipe from button stage,stipe from egg stage,stipe from elongation stage and stipe from maturation stage from strains Pyd15,Pyd21 and H1521,connected with the q-PCR results of nine specimens of mycelia,primordia,pileus from button stage,stipe from egg stage,pileus from egg stage,stipe from elongation stage,pileus from elongation stage,stipe from maturation stage and pileus from maturation stage of strain H1521 indicated:vv-rcd1 might play roles in egg stage,especially in the initial differentiation and growth period of pileus from egg stage of V volvacea.4.Results of Agrobacterium tumefaciens liquid-based PCR,double enzymes restriction and sequencing indicated that the forward,reverse and cycled fragments of RNAi of GME4331 and overexpression vector were successfully inserted into the vector pBHg-gpd,and obtained the Agrobaterium tumefaciens transformants with rcd1-RNAi and rcd1-overexpression vector,respectively.5.After two rounds identification for hygromycin resistence and hpt stability of putative transformants,we got 4 putative GME43310E and 3 GME4331RNAi transformants with stable hpt heredity.The result of qPCR showed that,compared with wild type strain H1521,the expression level of rcd1 of putative rcd1RNAi transformants 4i1-2,4i12-1 and 4i24.2 were down-regulated 3.41,5.14 and 1.56 times,respectively;the expression level of rcd1 of rcd1OE transformants 4010,405-2 and 4022 were up-regulated 1.84,1.20 and 1.15 times.The acquisition of rcd1-RNAi and rcd1-overexpression putative transformants indicated that the initial establishment of genetic transformation system of V.volvacea mediated by Agrobaterium tumefaciens.6.Biological property of the transformants showed that vv-rcd1 may not be relevant to the mycelial growth rate of V volvacea.The overexpression may cause the production of more chlamydospores and the accelerated senescence,which possibly indicated that the adaptation to outer environment of the rcdl overexpression tranformants weakened.In this research,we analyzed the structure and expression level of vv-rcd1,established the genetic transformation system of V.volvacea,then took preli minary investigation into the function of vv-rcd1 in the fuiting body formation and development process in V.volvacea through RNAi and overexpression methods.