Expression And Regulation Of CAZy Family Genes Of Volvariella Volvacea Grown On The Rice Straw As Well As During Stipe Elongation

Volvariella volvacea is a kind of typical straw-decay edible fungi as well as one of common mushroom vegetables.However,there are two problems restricting the development of straw mushroom industry:first,its biological efficiency is low;second,it is prone to open pileus and has a short shelf life.Its low biological efficiency is maily relative with the ability of degradating rice straw by V.vovacea,which carries out by the carbohydrate-active enzymes(CAZy enzymes).V.volvacea tending to open pileus was maily caused by rapid elongation of stipe in a short-term from the egg stage to the elongation stage,followed by the outer membrane splitting,the expanding pileus and less commodity value.The elongation of stipe is closely related to reconstruction of stipe cell walls,so CAZy enzymes(like glucanase and chitinase)are the final operators of stipe elongation.CAZy family genes are closely relative with the mycelium stage and the fruiting stage,which are two main stages in V.volvacea life cycle.Therefore,our research focused on the expression andregulation network of CAZy family genes of V.volvacea grown on the rice straw as well as during its stipe elongation.1.Primary establishment of expression and degradation model of V.volvacea CAZy family genes under the rice straw inductionSubstrates degradation is a complicated process.Our research mainly focused on several aspects:which part of CAZy family genes were up-regulated induced by rice straw;which part of CAZy enzymes were secreted extracellularly;how synergy works between different CAZy enzymes,enzymes and other assistant proteins;what transcription regulators specifically regulated these up-regulated CAZy genes are;what specific transporters for enzymes and nutrition moleculars in the whole degradation process are.We constructed a draft model of rice straw degradation through the identification of the main genes and enzymes working in the crucial steps of the degradation.The detailed contents were as follows:(1)Annotation and comparison of CAZy family genes in V.volvacea genomeWe obtained 540 CAZy genes in the whole genome of V.volvacea through the annotation.Of them,GH(Glycoside Hydrolase)family genes count for the most number(216 genes),AA(Auxiliary Activitie)family ranks second(110 genes).We made the cluster analysis based on the number of CAZy family genes though the comparison among 40 fungi with genome data,and the result showed large correlation between cluster and the roles of fungi in ecological system.V.volvacea and Coprinopsis cinerea fell into the same cluster,belonging to non-wood decay saprotroph,locating between white rot fungi and brown rot fungi.(2)Screening of CAZy genes for lignocellulose degradation in V.volvaceaWe screened 130 genes encoding cellulose and hemicellulose degradation enzymes and 78 genes encoding lignin degradation enzymes,respectively,based on the known activity and substrates for CAZy families.V.volvacea has the largest number of genes encoding cellulose and hemicellulose degradation enzymes among 40 fungi(ranking first),distributing in 16 CAZy families.AA9 family contains the most genes(32),followed by GH5 and GH10(20 genes,respectively).Enzymes in AA9 family belong to lytic polysaccharide monooxygenases,cleaving cellulose chains with oxidation;while enzymes in GH5 and GH10 degrade cellulose and hemicellulose by hydrolysis.(3)Multiple extracellular CAZy enzymes specifically secreted by V.volvacea induced by rice strawWe firstly screened substrates which induced high expression of CAZy enzymes(mainly focus on lignocellulose degradation enzymes)though extracellular enzymes activity determination.The results showed that rice straw had the strongest inducing ability among the 11 inducers.Further,we employed mass spectum to identify the secretory proteins in the fermentation broth of V.volvacea mycelia induced by rice straw.The results showed that there were 311 proteins specifically induced by rice straw,133 proteins of which were CAZy enzymes.And,AA9 family(21 proteins)and GH10 family(10 proteins)were in the majority in the 133 CAZy proteins,which were responsible for degrading cellulose and hemicellulose.(4)CAZy family genes of V.volvacea showed different expression patterns when induced by rice strawWe performed transcriptome and proteome sequencing aiming at the mycelia induced by rice straw,in order to determine the expression levels of V.volvacea CAZy genes.Transcriptional levels and translational levels of genes degrading cellulose and hemicellulose exhibited higher consistency by comparison.There were four types of CAZy genes degrading cellulose and hemicellulose according to their expression patterns:significantly up-regulated type(25 genes),consistently highly expressed type(16 genes),non-differentially lowly expressed type(86 genes)and significantly down-regulated type(3 genes).Next,we chosed 32 genes in AA9 family with the most cellulose degrading genes to confirm expression pattern by Q-PCR.The results among Q-PCR,transcriptome and proteome showed high consistency.Similarly,expression patterns of 32 genes in AA9 family falled into four above mentioned types:significantly up-regulated type(12 genes),consistently highly expressed type(3 genes),non-differentially lowly expressed type(15 genes),and one gene without expression level in transcriptome data.There was no down-regulated gene induced by rice straw in AA9 family.(5)Preliminary establishment of molecular mechanism model of V.volvacea mycelia degrade rice strawWe screened and identified several groups of genes and proteins in V.volvacea which specifically up-regulated expressed when induced by rice straw and had consistency in transcriptome,intracellular proteome and extracellular proteome.These groups of genes and proteins playing important roles in degrading rice straw included:(1)secretory extracellular CAZy enzymes(divided into:11 basically expressed type enzymes and 18 inducible up-regulated type enzymes);(2)membrane transport proteins(4 ABC transport proteins and 7 MFS transport proteins);(3)three specific transcription factors.At last,we constructed draft model of molecular mechanism of V.volvacea degraded rice straw based on those identified genes or proteins functioned in important steps of lignocellulose degradation2.Expression analysis and related metabolism regulation of some CAZy family genes during stipe elongation of V.volvaceaThe second part of our research was about the indentification of CAZy family genes involved in stipe elongation,as well as their expression levels analysis and metabolism regulation of stipe elongation in V.volvacea.Rapid stipe elongation is the most noteworthy feature during the fruiting body development of V.volvacea.Firstly,we screened CAZy family genes playing crucial roles in stipe elongation according to the digital gene expression profiles data,and chosed some of them to further clone,identify and analyze their detailed molecular function in the stipe elongation process.Next,we discovered the switch of metabolism activities as well as related regulation factors during the whole development of the V.volvacea stipe.We constructed a draft model of molecular mechanism of V.volvacea stipe elongation.The detailed contents were as follows:(1)Screening and identification of crucial CAZy enzymes genes during the stipe elongation of V.volvaceaWe obtained 16 CAZy genes significantly up-regulated in stipe elongation stage,according to expression profiles of different stipe development stages of V.volvacea.We chose exo-?-1,3-glucanase encoding gene(exg2),belonging to GH55 family,to further clone and detect its expression levels.The result revealed that the expression level of exg2 gene exhibited positive correlation with the elongation of stipe tissue.And the protein encoded by exg2 could hydrolyze glucan in cell wall and participate in the reconstruction of cell wall in stipe,thus playing a role as the final executive in the rapid stipe elongation.The other 15 genes include chitinase encoding gene,celluase encoding gene and ligninase encoding gene,and so on.Among these 15 genes,9 had predicted signal peptides or transmembrane structures,meaning that these 9 proteins may also work upon the cell wall and thus function in the stipe elongation through reconstructing cell wall of stipe.(2)Metabolism switch in stipe elongation process and preliminary construction of regulation mechanism model of V.volvacea stipe developmentThrough function enrichment of genes differentially expressed in the four development stages of V.volvacea stipe,we found activity of cell cycle pathway donw-regulated vastly from egg stage to elongation stage.Q-PCR was also performed to verify the expression levels of genes in cell cycle pathway and regulatory factors of cell division.It was confirmed that the rapid elongation of stipe was along with the weakening of cell division.We also identified in total 21 transcription factors regulating the metabolish activity switch and regulatory factors that could regulate the elongation of stipe cell.To summary,stipe elongation was the result of vertical elongation of stipe cell,accompanying with mitosis activity weakening in the rapidly elongating stipe cells.In conclusion,we constructed draft model of molecular mechanism of V.volvacea stipe elongation.