Functional Identification Of A Scaffold Protein PoxSte50 In Penicillium Oxalicum

Fungal scaffold protein Ste50 is closely related to the mitogen-activated protein kinase(MAPK)signaling pathway and plays an important role in cellular regulation.Ste50 acts as an adaptor protein to mediate membrane localization of MAPKKK Ste11 and forms a stable complex with Ste11,thereby mediating the MAPK cascade pathways,such as the pheromone reaction pathway and the hyperosmotic glycerol pathway.However,biological functions of the PoxSte50(Penicillium oxalicum Ste50)in P.oxalicum is poorly understood,especially in the regulation of expression of genes encoding plant-biomass degrading enzymes.In the previous work,gene PoxSte50 was deleted through homologous recombination technology in the P.oxalicum parental strain ?PoxKu70,to generate the deletion mutant ?PoxSte50.When directly cultured in liquid medium with crystalline Avicel as the carbon source for six days,the mutant ?PoxSte50 showed72% of reduction in the production of filter paper cellulase,compared with the?PoxKu70.Here this present thesis aims to study in depth the roles of PoxSte50 in expressional regulation of genes encoding plant-biomass-degrading enzymes(PBDEs)in P.oxalicum.Compared with the ?PoxKu70,the production of cellulase(filter paper cellulase,carboxymethyl cellulase,p-nitrobenzene-β-D-cellobiosidase),xylanase and amylase by mutant ?PoxSte50 decreased significantly,ranging from 35.6% to 98.4% after four days of Avicel or starch induction.By contrast,the p-nitrophenyl-β-glucopyranosidase production increased by 2.0–54.5-fold.The yields of cellulase,xylanase and amylase by the complementary strain CPoxSte50 had no significant difference from that by the?PoxKu70.Mutant ?PoxSte50 showed larger colony diameter and darker color on PDA,CM,glucose,or starch medium plates than the parental strain ?PoxKu70.Revesely,on Avicel plates,?PoxSte50 showed smaller colony diameter and lighter color.The spores produced by mutant ?PoxSte50 on PDA and glucose increased by 16.3–157%,while decreased by 83.8% on Avicel.There was no significant change on both starch and CM medium.Mutant strain ?PoxSte50 sporulates earlier on all the above medium plates.Mycelial accumulation of the ?PoxSte50 in glucose,soluble starch or Avicel medium is significantly lower than that of the ?PoxKu70,with a record of 8.8-52.5%,but in CM medium,did not affected.Transcriptomes of both strains ?PoxSte50 and ?PoxKu70 under Avicel induction for 24 h were sequenced.Comparative analysis indicated that a total of 2310 differentially expressed genes(DEGs)were obtained,with-1 ≤ |log2(fold change)| ≤1 and q-value ≤ 0.05 as the thresholds,including 82 PBDE genes,87 genes encoding transcription factor,19 kinase genes and 82 genes encoding sugar transporters.Fluorescence quantitative PCR analysis showed that the transcription levels of key cellulase genes(POX05587,POX04786,POX07535,POX01166 and POX06835)in the mutant ?PoxSte50 significantly decreased by 36.7–97.6% on Avicel for 12–48h,compared with that in the ?PoxKu70.The expression of two key xylanase genes POX06783 and POX08484 decreased significantly at 24–48 h,ranging from 76.5% to98.4%.The transcriptional levels of the key transcription factor genes known to regulate the expression of cellulase genes(such as POX01960/Clr B and POX02484)and kinase genes(such as POX00158/Fus3)showed a reduction of 50.2–91.3% at12–48 h.In addition,the cellodextrin transporter-encoding genes POX05915/Cdt D and POX06051/Cdt C at 4 h and 12 h increased their expression by 2.3–49.9-fold,while decreased by 64.9% and 82.2% at 24 h and 48 h,respectively.