Selection Of Differentially Expressed Transcription Factors And Functional Analysis Of Key Gene During The Morphogenesis Of Volvariella Volvacea

The Chinese mushroom, Volvariella volvacea (Bull.:Fr.) Singer, is a tropical and subtropical saprophytic fungus in the family Pluteaceae of the Basidiomycota. The lifecycle of V. volvacea is a complex process that involves a cascade of morphological events in fruiting body development. Several genes are responsible for regulation and control of the morphogenesis of V. volvacea fruiting body formation. In fact, the market value of V. volvacea was limited as difficult to storage and preservation that the short lifecycle resulted in the pileus-opening. The differentially expressed genes were obtained by comparing the gene expression level between two periods, from mycelia to primordia and from egg stage to elongation stage, respectively. Moreover, to identify the transcription factors of V. volvacea involved in primordia formation and stipe elongation, enrichment analysis of functional annotation for the expression profiles were performed. It could provide fundamental basis for functional verification and application in production that the preservation of V. volvacea was prolongated by using gene modification. The mainly results as follow:(1) In the present work,214putative transcription factors (TFs) belong to30transcription factor families were identified from the genome of V. volvacea via the pipeline of fungal transcription factor database (FTFD).(2) Compared the conservation of the TFs in different basidiomycetes and ascomycetes fungi, including V. volvacea, Schizophyllum commune, Laccaria bicolor, Coprinopsis cinerea, Ustilago maydis, Saccharomyces cerevisiae, Trichoderma reesei, Neurospora crassa, Aspergillus nidulans, by homology alignment. The results showed that62.6%transcription factors have homology with other eight fungi,37%is specific for V. volvacea, and46%is specific for basidiomycetes fungi. In addition, only16%transcription factors have homology in basidiomycetes and ascomycetes fungi. Therefore, the homology of transcription factors was low between basidiomycetes and ascomycetes fungi. However, the conserved domain of transcription factors among them is similar, although the whole amino acid sequences of transcription factors were not conserved.(3) To identify the differentially expressed genes in primordia and elongation stage,1503and1367differentially expressed genes were obtained by comparing the gene expression level between two periods, from mycelia to primordia and from egg stage to elongation stage, respectively. Of them,877and626genes were up-regulated and down-regulated expression from mycelia to primordia, respectively. Nevertheless, there have1004genes down-regulated expression, only363genes up-regulated, from egg stage to elongation stage. The enrichment analysis of functional annotation for the expression profiles of the developmental stages defined by mycelia, primordia, egg stage and elongation stage was performed. Functional terms involved in regulation of cellular process and metabolic process, were over-represented in genes up-regulated of primordia. Genes involved in cellular component biogenesis, anatomical structure formation and multicellular organismal process were enriched in the group of genes down-regulated of primordia. However, functional terms involved in regulation of cellular component organization and of cellular process were enriched in the down-regulated genes of elongation stage. Genes encoding anatomical structure formation, multi-organnism process were enriched in the group of genes up-regulated of elongation stage. The enrichment analysis of functional annotation illustrated that the regulation of gene expression played a crucial role in the formation of fruiting body based on the different expression analysis. We found that14%differentially expressed TFs belong to homeobox family in primordia and elongation stage. It may be paly more important role in these processes.(4) By the differential expression analysis as above mention,8differentially expressed homeobox TFs were obtained, including5up-regulated expressed in the primordia and3down-regulated expressed in the elongation stage of V. volvacea. In fact, quantitative real-time PCR (qRT-PCR) was used to analyze gene expression value in mycelia, primordia, egg stage, elongation stage. The results showed that the gene expression levels of five genes (GME10392, GME1763, GME860, GME11623, and GME8409) were higher in primordia than in mycelia, whereas the gene expression levels of three genes (GME918, GME8013, and GME2319) in elongation stage were lower than in egg stage. The qRT-PCR results were consistant with the gene expression profile, and inferred that these differential expression homeobox TFs may contribute to morphological formation of the fruiting body of V. volvacea.(5) The molecular cloning and sequencing results of PCR products illustrated that the sequences of GME918(GenBank accession numbers:JX843776) from strains PYd15and PYd21were identical. About26million high-quality reads and2,408,000,220nt nucleotides from transcriptome sequencing. Of them,3,632reads could map to the reference sequence in analysis of gene structure of GME918. GME918spanned2856bp including an open reading frame of1149bp, and composed four exons and three introns. In addition, the alternative splicing in the first intron was found in studying the gene structure.(6) RNAi vector of GME918was constructed according to its conserved domain. It was evaluated by using polymerse chain reaction (PCR) and restriction enzyme digestion. Moreover, it was transform to V. volvacea strain H1521by Agrobacterium tumefaciens. The results showed that the colonial morphology and mycelial growth rate of transformant changed significantly. Therefore, the results indicate that transcription factor GME918could regulate the colonial morphology and mycelial growth rate of V. volvacea.