Regulation Of Htf And Stua On Sporation And Glycoside Hydrolases Expression In Penicillium Oxalicum

In Filamentous fungi, conidia formation provides a mechanism undergoing asexual propagation and endowing dormancy to avert from rugged surroundings. Hyphae starts to extends upon the substrates and secretes large amounts of hydrolases to uptake nutrients once conidiophore germinates. Conidia formation and hydrolases expression are under the control of the systemic, complex, equisite network of signal transduction and transcriptional regulation. In order to meet with era of the ethanol biofuels, cellulase, currently the third largest industry enzyme worldwide is marching forward to the largest volume enzyme with the deeper development of knowlegement and technology about filamentus fungi. Long-term strain improvements of the Penicillium oxalicum opened the way for industrial cellulase production. Genome sequence comparision significantly shows the transcriptional factors enrichment for the mutated proteins. System establishment.of transcriptional factors deleted collection and funtional analysis showed that some new transcriptional factors took part in conidiation and lignocellulose-degrading enzymes synthesis. In this collection, two transcriptional factors involved in the conidiation and carbon source metabolism received further investigation. A better understanding on the regulation mechanism of the growth development and lignocellulose degradation in Penicillium oxalicum, with further investigation transcriptional factor function, is full of theoretical meaning and applicable value.The main results of the research are as follows:1Characterization of transcriptional factor Htf in Penicillium oxalicum.Phylogenetic analyses showed that transcriptional factor Htf（PDE₀7199） is an orthologue protein of Homeodomain protein. There was no conidiophore structure variation other than severe lag of conidiation in the htf deleted strain cultured on various carbon sources. Conidiation lag could be overcome by recompleted the deleted mutant strain. Through the transcriptional profiling, conidiation lag couldbe almost attributed to the dramatically descent of brlA transcriptional level. Meanwhile, the homeodomain protein played an important roles in carbon starvation response and cellulose induction. The hydrolyzing ring of the htf deleted mutant strain cultured on microcrystalline cellulose plate was bigger than that of the wide type. In liquid fermentation with microcrystalline cellulose induction, the htf deleted mutant strain increased0.53times of FPA,1.43times CMCase,0.54times cellobiohydrolase,0.36times β-glucosidase activities and to some degree of the transcriptional level of cellulase gene cbhl, egl, bgll and bgl2. There was no effects on growth but causes decreased cellulase activity in the htf overexpression strains. Also, in the overexpression strains, the transciptional level of cellulase genes had a negative relationship with the transcriptional level of htf gene.2Characterization of transcriptional factor Stu A in Penicillium oxalicum.According to the phylogenic analyses, the transcriptional factor StuA （PDE₀4095） from P.oxalicum was supposed to belong to the APSES family, which is restricted to fungi. Deleting the stuA gene results in growth impairment with the presence of light when the strain cultured with Mendal’s salt. A Vogel’s salt couldrecover vitality, however,the strain failed to conidiation any more regardless of the carbon source and nutrient. Moreover, no conidiophore was in the microscopic detection. brlA, a key regulator of the central pathway regulating conidiation, was blocked in the transcriptional level while flbC, an upstream modulator was not directly suffered from the stuA gene deletion. It may be concluded that stuA is an upstream core activator of brlA and acted as an on-off switch regulator of the conidiation pathway in P.oxalicum, with the additional fact that six analogous binding motif for the APSES protein closely arranged on the brlA promoter from-540bp to-950bp. It was also noteworthy that no hydrolyzing ring on the microcrystalline cellulose plate and smaller hydrolyzing ring on the amylum plate when the stuA deleted mutant strain compared with the wide type. Enzyme analysis for the supernatant of liquid fermentation demonstrated that stuA knocked-out brought about increasing FPA, xylanase, CMCase activities and decreasing cellobiohydrolase, β-glucosidase activities. Under the response of carbon starvation, cbhl, egl and amyR enhanced transcription while amylase15A reduced transcription. However, all the four genes showed declined transcription level when the deleted strain induced by the microcrystalline cellulose. It could make a conclusion that StuA participated in the process of cabon starvation response, amylase gene expression and cellulase gene induction. 3Compilation of the DGE（Digital Gene Expression Profiling） data of the stuA deleted strainâ–³stuA.Analysis on the some770different expression genes betweenâ–³stuA and wild type induced with microcrystalline cellulose revealed that StuA is a wide-scope regulator in P.oxalicum. Apart from the regulation in conidiation and glycosidase expression, StuA, to some extent, imposed effect on the genes involved in cell metobolism, cell cycle and apoptosis, cell signal transduction, cell transportation.