Functional Identification Of The Two-component Regulatory System Genes In Penicillium Janthinellum GXCR

Copper(Cu)are the nutrient elements of organisms.Low concentrations of copper(Cu)promote the growth of organisms.High concentrations of copper have an adverse effect on the growth and survival of organisms.Fungi have a stronger anti-copper effect,but little is known about its anti-copper mechanism.A highly copper-resistant filamentous fungus strain--Penicillium janthienllum GXCR was screened in this laboratory.This high resistance makes Penicillium janthienllum GXCR a potential strain to deal with ecological heavy metal pollution.In order to make the strain better play the function of dealing with heavy metal pollution,or to transform the strain into a more functional engineering strain,it becomes very critical to understand the high resistance of Penicillium janthienllum GXCR at the molecular level.The cascade reaction of SLN1-YPD1-SSK1 is the most classical two-component system with the most effective osmotic regulation in saceromycetes.The histidine kinase(SLN1)senses the environmental pressure and self-phosphorylates,then transfers the phosphate group to the phosphate transfer protein(YPD1),and then transfers the phosphate group to the response regulatory protein(SSK1).Thus,MAPK cascade reaction is activated to cope with the extracellular environmental pressure.Slnl-ypdl-skn7 is another branch of the two-component regulation system of s.cerevisiae,which is mainly involved in oxidative stress and cell wall synthesis of s.cerevisiae cells.In this study,based on transcriptome data and comparative analysis of its homologous sequences,the ORF sequences of the genes homologous to the genes of the two-component regulatory system of s.cerebrae were obtained in the CDS sequences of Penicilliumjanthienllum GXCR,and were named PjSKN7,PjSSK1,PjYPD1,PjSLN1,PjSLNl-1 and PjSLN1-2,respectively.In this experiment,the genes of GXCR two-component regulatory system of Penicillium janthienllum GXCR were successfully cloned,and the PjSKZN7,PjSSKl,PjYPD1,PjSLN1,PjSLN1-1 and PjSLN1-2 were expressed heterogeneously in s.cerebrae.PjSKN7 has obvious anti-copper effect.When the content of activated oxygen and proline in the cells of s.cerevisiae mutant under the stress of heavy metals was detected,it was found that the mutant of s.cerevisiae PjSKN7 had lower content of reactive oxygen and higher content of proline.Real-time fluorescence quantitative PCR was used to detect the expression levels of the two-component regulatory genes of Penicillium janthienllum GXCR under the stress of high concentrations of heavy metals and salts:PjSKrN7,PjSSKl,PjYPD1,PjSLN1,PjSLN1-1 and PjSLN1-2.The results showed that the expression of two-component genes of penicillium microphylla was generally expressed as stage expression.PjSKN7,PjYPD1,PjSLN1,PjSLN1-1 and PjSLN1-2 were up-regulated under heavy metal stress,while PjSSK1,PjYPD1,PjSLNl,Pj SLN1-1 and PjSLN1-2 were up-regulated under high concentration salt stressThis study indicated that the genes PjSKrN7,PjYPDl,PjSLNl,Pj SLN1-1 and PjSLN1-2 of Penicillium janthienllum GXCR were involved in the anti-copper mechanism.PjSSK1,PjYPD1,PjSLNl,PjSLN1-1,Pj SLN1-1 participate in the osmotic regulation mechanism.This study provides molecular basis for further study on the high anti-heavy metal properties of Penicillium janthienllum GXCR and its application in the treatment of ecological heavy metal pollution.