Study On Differential Gene Expression Of Homokaryon And Heterokaryon Of Volvariella Volvacea

Now, among more than20kinds of mushroom that widely cultivated in China,the production of straw mushroom [Volvariella volvacea (Bull.ex Fr.) Sing.] waseighth which means it has important economic values. The results of V.volvaceacrossbreeding indicated that strains fruiting were heterokaryon which could be stablyinherited. However, the molecular mechanism of its formation is still blank. A furtherstudy on the molecular mechanisms of heterokary formation can help us reveal theinheritance of V.volvacea and guide molecular breeding of V.volvacea. It hasimportant theoretical significance and potential applications. Previously, we have gottwo single spore isolates named PYd15and PYd21respectively from PY which wasone of main cultivars in Fujian province. A hybrid H1521was got by mating PYd15and PYd21. Results of molecular marker detection showed that H1521which hadgenetic material of PYd15and PYd21was a heterokaryon. In this study, using PYd15,PYd21and H1521as materials, the differences between homokaryon andheterokaryon of V.volvacea on transcriptome and proteome scales were studied. Andthis will lay a theoretical foundation on formation of heterokaryon in V.volvacea. Themain research contents of this thesis are concluded as follows.(1) The genome of PYd21was de novo sequenced by Solexa genome analyzertechnology, and then sequencing reads were assembled by Soapdenovo software.Ultimately, a number of302Scaffold were got, and the genome of PYd21was37200239bp. The longest and shortest Scaffold were1845736bp and1003bprespectively. By Augustus(version2.1) software used for prediction of fungi gene, wegot11534genes and total length of it was16837461bp which accounting for45.26%of the entire genome. The average length of genes was1459bp, and GC content ofgenes were50.86%. Results of gene annotation showed that there were completemetabolic pathways about glycolytic pathway. From glucose-6-phosphate to pyruvate,there were10enzymes annotated by11genes of V.volvacea.(2) Using Solexa Genome Analyzer platform, a transcriptome containing equivalent mRNA of8samples including mycelium of PYd21, PYd15and H15-21,primordium, stipes of fruiting bodies of button, egg, elongation, and mature stages,was sequenced. And after sequencing, assembly, annotation and bioinformaticanalysis were carried out. At last, we got a number of34287Unigene with the averagelength557nt. After being aligned with COGs database, a total of12666Unigene wereannotated involving25functional categories. Using KEGG database, we annotated9300Unigene which involved in a total of105pathways.(3) Using Solexa Genome Analyzer platform, digital gene expression profilesfor PYd15, PYd21and H1521were conducted. After sequencing, all Clean Tags werealigned to reference gene database obtained from (1) and (2), differently expressiongenes between homokaryon and heterokaryon were analysis by informatics methods.A total of5753854,5701781and5950904Clean Tags that corresponded to86772,85626and106250Distinct Tags for PYd15, PYd21and H1521respectively.To the end, we obtained13667,14387and13780tag-mapped sense transcripts forPYd21, PYd15and H1521library respectively. By a rigorous algorithm method, weobtained2053and4657differentially expressed genes which was the molecular basisof difference for homokaryon and heterokaryon among PYd15vs H1521and PYd21vs H1521. Gene ontology(GO) functional enrichment analysis showed that11significant terms were enrichmented between PYd15and H1521, and no term wasenrichmented between PYd21and H1521. Pathway enrichment analysis showed that4most meaningful pathways between PYd15and H1521were enrichmented, and nopathway was enrichmented between PYd21and H1521.(4) Using isobaric tags for relative and absolute (iTRAQ)-coupled2DLC-MS/MS technologies, differential proteome for homokaryon and heterokaryonmycelis were studied. Different expression proteins between homokaryon mycelia andheterokaryon mycelia were identified and analysis by bioinformatics software. To theend,1039proteins containing2335unique peptides were identified, and of this,1030proteins had quantitative information. There are45different proteins betweenhomokaryon and heterokaryon mycelis, of which23proteins were up-regulated and22were down-regulated in heterkaryon. (5) Structure and expression levels among PYd15, PYd21and H1521ofglycolytic genes were studied. Based on genome sequence of PYd21,7genes ofglycolytic pathway from both PYd15and PYd21were cloned and sequencd,alignment revealed that7genes sequences were identical in the two strains, and therewas no polymorphism. Using reads database of transcriptome, structure analysis ofglycolytic pathway genes showed that the length of genes was from1015bp to3494bp, the number of introns was from5to13,7genes had alternative splicing type ofintron-rentention(IR) and3genes had alternative splicing type of alternative3, splicesite(A3SS) during RNA processing. Using digital gene expression data, expressionleves of glycolytic pathway genes in PYd15, PYd21and H1521was studied and theresults showed that except phosphoglucomutase (PGM) gene, expression levels ofother genes in heterokaryon were higher than in homokaryon. Expression level ofglycolysic pathway genes in PYd15, PYd21and H1521were tested by real-timequantitative PCR, the result showed expression levels of all genes in heterokaryonwere higher than in homokaryon and gene expression levels had synergistic effect inheterokaryon. The expression levels of glycolysic pathway genes has a significantpositive correlation with growth rate of mycelium which indicated that the two typesnuclei in H1521interacted each other, and this made the expression levels ofglycolysic genes increase, the latter accelerate growth rate of mycelium.