| Penicillium oxalicum HP7-1 is a soil filamentous fungus with complete cellulase system.Previous studies have found that Penicillium oxalicum MAP kinase(mitogen-activated protein kinase,MAPK)plays an important role in regulating the production of cellulase and xylanase.Through gene annotation of Penicillium oxalicum HP7-1 genome,44 genes involved in the MAPK pathway were screened.Among the 44 genes,one gene was annotated to encode phospholipase.Phospholipases play an important role in the immune and inflammatory responses of mammals and plants,mainly in the growth and development of mammals and physiological regulation,while in plants they are related to stress resistance and stress response of plants.However,research on the role of phospholipase in fungi is still limited.Therefore,the function of phospholipase in Penicillium oxalicum was further explored.In this study,the predicted phospholipase genes were screened and their biological functions were studied in Penicillium oxalicum.Among the 10 predicted phospholipase genes,the deletion of a single gene of nine genes in Penicillium oxalicum resulted in various degrees of changes in fungal growth,spore number,cellulase and xylanase production.Among them,the gene POX07277 encodes phospholipase PLA2.After a shift cultivation,the production of cellulase and xylanase of the mutant ΔPOX07277 decreased by 22.5-82.8%(filter paper cellulase,carboxymethylcellulase and p-nitrophenyl-beta-cellobias e),while the production of p-nitrophenyl-beta-glucopyranosidase increased by 5.8-127.8%.Therefore,this mutant was selected for further study.RT-qPCR analysis showed that POX07277 is dynamically involved in the expression of cellulase gene and xylanase gene under the induction of Avicel.At 12 h after a shift cultivation,the expression of cellulase genes POX06835 and POX05587 increased by 100.26% and 827.32%,respectively;the expression of cellulase genes and xylanase genes POX04786,POX01166,POX05571,POX07535,POX06783 and POX08484 decreased by 29.89-61.47%,respectively;at 24 h after a shift cultivation,the expression of cellulase genes and xylanase genes POX05587、POX04786、POX01166、POX05571、POX07535、POX06783 and POX08484 decreased by 52.55-93.43%,and the expression levels of cellulase gene POX06835 increased by 395.91%,respectively.At 48 h after a shift cultivation,the expression of cellulase genes and xylanase genes POX06783,POX08484,POX05587,POX04786,POX01166,POX05571,POX07535,POX06783 and POX08484 decreased by 76.22-91.61%,respectively;the expression of cellulase gene POX06835 increased by 94.14%.Therefore,POX07277 may be involved in the expression of cellulase genes and xylanase genes through intracellular metabolic regulation or potential signaling pathways.The colony phenotype observation,spore number analysis and hypha observation analysis of the constructed complementary strain CPOX07277,mutant strain ΔPOX07277 and parental strain ΔPoxKu70 showed that the phospholipase gene POX07277 is involved in the production of hyphae and spores.Compared with the cultivation of the mutant ?POX07277 without the addition of arachidonic acid(AA),the yield of cellulase and xylanase of the mutant ?POX07277 increased by 60.7-258.6% when the final concentration of 0.2 mg/m L AA was added to the culture medium,and the yield of cellulase and xylanase in mutant ?POX07277 increased by 39.4-218.4% when the final concentration of 0.4 mg/m L AA was added to the culture medium.Compared with the parental strain ?PoxKu70 cultivated without AA,the cellulase and xylanas e production of ?PoxKu70 increased by 30.4-144.28% when the final concentration of 0.2 mg/m L AA was added to the culture ?PoxKu70,and decreased by 28.2% when the final concentration of 0.4 mg/m L AA was added.Low concentration of exogenous supplementation of AA increased the production of cellulase and xylanase by the mutant strain ?POX07277,while high concentration of exogenous supplementation of AA inhibited the production of cellulase and xylanase.The effects of AA on the expression of cellulase genes and xylanase genes in?PoxKu70 and ?POX07277 were examined by RT-qPCR,respectively.The results showed that the transcription levels of all cellulase genes and xylanas e genes were inhibited by 0.4 mg/m L AA in the pre-culture period(12-24 h)of the strain ?PoxKu70 compared with those without AA,and the gene expression was reduced by 50.4%-95.1%.After 48 h of cultivation,the expression of cellulas e genes POX04786,POX01166,POX05571,POX07535,POX06783 all significantly increased at 75.1-564.7%.Compared with the gene transcription level without AA,when the mutant ?POX07277 was supplemented with 0.4mg/m L AA in the culture medium,the transcription levels of all tested cellulas e genes and xylanase genes significantly decreased by 35.2-69.5% at 12 h of cultivation,except for the cellulase gene POX07535.At 24 h of cultivation,the expression levels of cellulase and xylanase genes POX04786、POX06783 and POX06783 significantly decreased by 15.8-54.7%,while the expression levels of cellulase gene POX07535 significantly increased by 159.6%.At 48 h of cultivation,except for cellulase genes POX01166 and POX06835,the transcription levels of all tested cellulase genes and xylan genes significantly increased by 78.7-494.8%.These data suggest that AA can compensate for the effect of POX07277 deletion on cellulase gene and xylanase gene expression in Penicillium oxalicum.The expression of POX07277 is regulated by the known transcription factor PoxCxr B,which positively regulates the expression of cellulase gene and xylanase gene in Penicillium oxalicum.In the late stage of mutant ?PoxCxr B cultivation(12-48 h),the expression of phospholipase gene POX07277 increased by 60.6-254.7%.For the parental strain ?PoxKu70 with addition of different concentrations of AA,with the increase of AA concentration and cultivation time,the transcription level of the transcription factor gene PoxCxr B gradually increased,indicating that AA can induce the expression of the transcription factor gene PoxCxr B in Penicillium oxalicum.This study preliminarily identified the function of the Penicillium oxalicum phospholipase gene POX07277 and proposed a model related to the involvement of cellulase and xylanase gene expression with phospholipase gene POX07277.These findings contribute to the understanding of fungal phospholipase function and provide new insights into the regulation mechanism of fungal cellulase gene and xylanase gene expression. |
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