The Expression Analysis Of B Mating-type Factor And Its Downstream Genes Related With MAPK Signal Pathway In Volvariella Volvacea

Volvariella volvacea(Bull.:Fr.)Sing.,also names straw mushroom,is a kind of important edible fungus growth in tropical and subtropical.In 2013,the genome of two single-spore isolates V23-1 and PYd21 from the main cultivation strain V23 and PY1 were sequenced and published articles respectively.In this study,we analyzed the B factors of V.volvaceca deeply based on the genome,DGE(Digital Gene Expression Profiling)and transcriptome data.At the same time,we constructed its downstream of MAPK signal pathway,screened of important genes and expressed in V.volvacea.The main results are as follows:(1)Downloaded the homologous pheromone receptors of other basidiomycete fungi and then found that there are four pheromone receptors in the genome of PYd21 by homologous comparison method of bioinformatics.Three of them have seven transmembrane structures,while the other one contains only five transmembrane structures.In v.volvacea STE3.1 upstream also found a pheromone precursor,length of 47 amino acids,containing conservative "CAAX" and "ER" motif.DGE data and fluorescence quantitative PCR showed that the expression level of these four pheromone receptor gene increased sharply in primordium stage.(2)We constructed the MAPK signaling pathways downstream of the B mating-type factor in V.volvacea through the analysis on the annotation data from the genome,transcriptome data,combined with the bioinformatics analysis.The DGE data demonstrated that all 16 genes related with MAPK signaling pathway expressed lowly,but two genes VvSte20 and VvSte7 two genes expressed highly and increased sharply in primordium stage.The result of fluorescence quantitative PCR further verified.Phylogenetic tree showed that the two genes encoding conservative proteins were homologous with other basidiomycete fungi of homologous proteins.(3)After analyzing the structures of VvSte20 and VvSte7,we used the exon 4 of VvSte20 as the fragment for long-term interference,the fourth introns as the loop in the RNAi fragment.And then ligated the RNAi fragment into the vector pBHg-VvSte20 RNAi with the traditional enzyme digestion and ligation methods.For VvSte7,we used overlap extension PCR method to ligate the fourth exon as long-term interference fragment,the fourth introns as the loop.Then a RNAi vector for VvSte7 pBHg-VvSte7 RNAi vector was constructed.At the same time,with the traditional enzyme digestion and ligation methods two overexpression vector pBHg-VvSte20 OE vector and pBHg-VvSte7 OE vector were constructed.(4)Transformed the four expression vectors into Agrobacterium GV3101.Then transformed them into V.volvacea by the method of Agrobacterium-mediated respectively.Pick out the putative transformants of V.volvacea which could still grow on PDA containing hygromycin and cefotaxime antibiotics plates after initial and second screen.After 5 generations culturing on PDA,genome DNA of putative transformants were extracted.Then we detected the hpt genes and the purpose gene fragment using PCR amplication.At the end,we got four RNAi transformants and one overexpression transformants of VvSte20 gene,three RNAi transformants and two overexpression transformants of VvSte7 gene.qRT-PCR was performed to detect the expression of purpose gene,and the result showed that the gene expressed down-regulated in mutants of RNAi while up-regulated in mutants of overexpression,which further confirmed that they were real transformants.(5)Detection the biological characteristics of mutants showed that in the RNAi mutants they grew slower significantly in PDA plate,while in the overexpression mutants,growth velocity difference is small,but fast chlamydospore formation,growth in the cultivation bag also shows the result.Put the mutants to get fruiting-body respectively and the results were as follows:First,put the RNAi mutants to get fruiting-body test on bed cultivation.The results demonstrated that the RNAi20-10,RNAi7-9 could not get any fruiting-body in three repeats;the RNAi7-D mutants product only one fruiting-body in one test of three repeats.The mutants of RNAi20-4,RNAi20-21 and RNAi7-1 could product the fruiting-body,but the size of fruiting-body was small.The mutant of RNAi20-5 almost is the same with wild type(WT)strain.Second,put all mutants to get fruiting-body test on cultivation bag.The mecelium of RNAi7-D and RNAi7-1 from VvSte7 couldn't grow at cultivation medium;The mycelium of RNAi7-9 grew normal but couldn't get fruiting-body;The four RNAi mutants of VvSte20 could grow normal mecelium while couldn't get fruiting-body except RNAi20-4.But the mushroom of RNAi20-4 developed slowly and stained at button stages;In the three overexpression mutants,all of them could get fruiting-body.The yield OE7-1 and OE7-3 was higher than wild type(H1521),and the size of fruiting-body was also bigger than wild type(H1521);The time of pileus opening of three overexpression mutants all delayed;The mutants of OE20-15 was almost the same with wild type(WT)strain.(6)Detect the expression level of related genes in mutants of VvSte7,the results showed:in three RNAi mutants,the expression level of genes downstream of VvSte7 significantly suppressed,and the genes upstream of VvSte7 also showed the cut;The two overexpression mutants showed different:in OE7-1 mutant,genes upstream and downstream of VvSte7 are presented to raise the level of gene expression,while in OE7-3mutant,only VvSte7 and its downstream gene MAPK(GME7452_g)characterized by trace increases,other genes expression were down-regulated.Therefore,VvSte7 and VvSte20 as genes of MAPK signaling pathways,they are related with mycelium growth,chlamydospore formation in V.volvaceo.These results in this study will provide important data to support in-depth studying the molecular mechanism of forming fruiting-body in V.volvacea and other edible and medicinal mushroom.