| As a renewable energy,bioenergy has a great potential application.Low-cost of renewable plant biomass are used as the excellent feedstock for bioenergy production via biorefinery.Hydrolysis and saccharification of plant biomass is the key technological bottleneck limiting biorefinery idustrialization,mainly due to low production and high cost of plant-biomass-degrading enzymes such as cellulase and raw-starch-degrading produced by fungi.How to improve the yield of fungal plant-biomass-degrading enzymes have been become a research hotspot so far.In-depth understanding of regulatory mechanism of the expression of genes encoding fungal plant-biomass-degrading enzymes has a great significance of genetic engineering for improving the production of plant-biomass-degrading enzymes.The mitogen-activated protein kinase pathway plays an important regulatory role in cellular processes.Previous work identified 44 genes involved in the mitogen-activated protein kinase(MAPK)pathway through screening the annotated genome of Penicillium oxalicum wild-type strain HP7-1.In present thesis,18 of 44 genes were successfully knocked out in the parent strain?Pox Ku70.The production of filter paper cellulase(FPase)was measured,when the constructed deletion mutants directly cultivated for six days.A mutant?POX07071 showed 86.2% of reduction in FPase production compared with the parental strain ?Pox Ku70.POX07071 was rename as Pox Ste18 and conducted further research.Enzymatic activity tests after a transfer revealed that the cellulase and xylanase production of the deletion mutant ΔPox Ste18 cultivated in Avicel medium for four days after a transfer from glucose,decreased by 20.1–96.1%,and the amylase(raw cassava starch-degrading enzyme and soluble starch-degrading enzyme)production decreased by 73.5–76.6% when in soluble starch medium.The enzymatic production of the complementary strain CPox Ste18 returned to the level of the parental strain ?Pox Ku70.Compared with the the parental strain ?Pox Ku70,the colony size and culture color of the mutant ΔPox Ste18 became irregular and lighter,when cultivared on plates containing glucose,PDA,Avicel or soluble starch medium,insertion observation revealed that the mutantΔPox Ste18 had sporulation time earlier than ΔPox Ku70.Both the number of spores and mycerial accumulation produced by the mutant ΔPox Ste18 showed reduction,when cultivated in either glucose or Avicel medium.Comparative analysis of transcriptomes from the mutant ΔPox Ste18 and the parental strain ?Pox Ku70 cultivated for 24 h after Avicel induction revealed1329 differentially expressed genes(DEGs)using |log2 fold change| ≥ 1.0 and Probability ≥ 0.8 as the thresholds.Of them,739 DEGs were up-regulated(1.0≤ log2 fold change ≤ 11.9)and 590 DEGs were down-regulated(-7.8 ≤ log2 fold change ≤-1.0).Notably,41 DEGs were annociated as predicted transcription factors,20 genes(1.0 ≤ log2 fold change ≤ 5.0)were up-regulated,and 21 genes(-3.3 ≤ log2 fold change ≤-1.0)were down-regulated,of which five genes had been reported to be involved in the regulation of cellulase production.54 genes were involved in cell wall degrading enzymes genes encoding cell wall degrading enzymes are 54(48 genes are up-regulated and 6genes are up-regulated),including 2 key cellobiohydrolases(POX04786/Cel6 A and POX05587/Cel7A-2),6 xylanase genes(POX00063,POX03430,POX04274,POX05916,POX06601 and POX06783/Xyn11A),8 endocellulases(POX01166,POX01896,POX04137,POX05570,POX05571/Cel7 B,POX06147,POX06983 and POX07535)and 5 β-glucosidase genes(POX00968,POX03062,POX03642,POX006079 and POX0006835/Bgl1),these genes were down-regulated.In addition,two amylase genes POX01356(Pox GA15A)and POX03741 are involved,both of which are down-regulated genes(-2.4 ≤ log2 fold change ≤-1.0),where POX01356(Pox GA15A)and POX03741 are involved,both of which are down-regulated genes(-2.4 ≤ log2 fold change ≤-1.0),where POX01356(Pox GA15A)encodes raw starch saccharification enzyme.In addition,one gene was known to be involved in the regulation of amylase production.Real-time quantitative PCR(RT-q PCR)analyses indicated that the transcription levels of the raw-starch-degrading enzyme gene POX01356/Pox GA15 A,glucoamylase gene POX02412,α-amylase gene POX09352/Amy13 A and their regulatory gene POX03890/Amy R were down-regulated by 18.0-85.6% in the mutant ΔPox Ste18 in comparison with that of the parental strain ΔPox Ku70 at 24 h after soluble starch induction.In addition,the expression of two cellobiohydrolase genes POX05587/Cel7A-2 and POX04786/Cel6 A,two endo-β-1,4-glucanase genes POX01166/Cel5 B and POX05571/Cel7 B,one β-glucosidase gene POX06079 and one endo-xylanase gene POX06783/Xyn11 A showed the 31.0–73.2% of reduction at 24 h after Avicel induction.Moreover,the Pox Ste18-overexpression strain OEPox Ste18 was constructed.Compared with the parental strain ?Pox Ku70,the colony size of the OEPox Ste18 became large on plates containing glucose,PDA,or Avicel medium.The production of FPase,CMCase,p NPCase and xylanase of the OEPox Ste18 decreased by 36.3–89.1%,whereas p NPGase production increased by 5.0–6.6 folds.RT-q PCR analyses displayed that the expression of the major cellulase gene and xylanase gene decreased significantly in the OEPox Ste18 compared with the parental strain ?Pox Ku70,while the transcription level of the Pox Ste18 increased significantly.Transcriptome values are consistent with quantitative data. |
PDF Full Text Request