Cloned And Analysis Of Diffetential Expression Of Three New Mnp Genes From Cerrena Unicolor

White-rot fungi have an ability to degrade lignin and aromatic compounds via secreting various extracellular isoforms enzymes.The major extracellular isozymes included Laccase(Lacc,EC 1.10.3.2),Lignin Peroxidase(LiP,EC 1.11.1.14)and Manganese Peroxidases(MnP,EC 1.11.1.13).Environmental pollution is becoming a more and more serious problem in the modern society,and the use of white rot fungi and their enzymes are becoming more and more popular in environmental governance.MnP has an important role in lignin degrading enzymes,furthermore,MnP has the characteristic of extensive substrate diversity,and it has also been widely studied and applied.As the limit of strain and environment,the applications of white rot fungi and their lignin degradation enzymes are difficult.Hence,it is a premise to find new strains and understand the mechanism of lignin degrading enzymes for the applications of white rot fungi and their enzymes in environmental pollution.In this paper,the strain named Cerrena unicolor was identified from the nature by the technology of Morphology and Molecular biology,and the decolorizing ability of dyes and secretory ability of MnP by C.unicolor were determined.Then genes of mnp isozymes from C.unicolor were cloned and analysised.On this basis,by Fluorescence quantitative PCR,the difference of expression among different genes of mnp isozymes from C.unicolor under various treatments was analysised.These results established the foundation of elucidating the Mechanism of mmp genes in the future.The main results were as follows:1.Isolation and purification Mycelium of C unicolor growing in rotten wood on Changbai Mountain,then the microscopic observations and biological characteristics of purification mycelium were studied,and the results suggested that the microscopic observations and biological characteristics of purification mycelium were consistent with features of C.unicolor.Furthermore,ITS sequence of C.unicolor CB1 was cloned,On this basis,a phylogenetic tree for ITS sequences of 16 different C.unicolor stains include strain CB1 was established,and the results revealed that there was a greater similarity between strain CB1 and other strain,and they could be belong to the same group.According to comprehensive analysis,the test strain CB1 belong to C.unicolor,was named Cerreona unicolor CB1(hereinafter referred to as stain CB1).2.After strain CB1 identified,the strain was used to study the ability to degrade dyes(Reactive Black and Reactive Red)under solid conditions.The results revealed that stain CB1 had an good ability to degrade the two dyes under solid conditions,and the decolorization rate of 50 mg·L-1 Reactive Black reached 100%on day 10,and 100 mg·L-1 Reactive Red reached 100%on day 12,even if high concentration of dyes,500 mg·L-1 Reactive Black and Reactive Red,the decolorization rate were more than 50%on day 12.On this basis,the degradation ability of the strain CB1 to dyes(Reactive Black and Reactive Red)under liquid conditions were systematically studied.The result revealed that Reactive Red could be more easily degraded by strain CB1 than Reactive Black,and there was obvious inhibition effect by 250 mg·L-1 Reactive Black in the process of degradation,but it was not obvious to 500 mg·L-1 Reactive Red.For degradation of Reactive Black and Reactive Red by strain CB1,the best carbon source was fructose and glucose,respectively.The best nitrogen source was urea and ammonium nitrate,respectively.The best pH values were both pH=5,and the best concentrations of Cu2+and Mn2+were both 0.1 mmol·L-1,and The best inoculation quantity were both 7 fresh mycelium discs with diameter 8 mm.3.To investigate secretion ability of MnP and application prospect of MnP from C unicolor,the curve of secretion MnP was determined under based conditions of lignin degrading enzymes from C.unicolor CB1,the results revealed that the MnP activity could be observed after day 3 from C.unicolor CB1.and peaked on 7 day,and then MnP activity reduced volatility.After determining the optimal time of secreting MnP by C.unicolor CB1,then we optimized the culture conditions of producing MnP.The result showed that the optimal conditions of secretion MnP from C.unicolor CB1 were Fructose 10 g·L-1,Ammonium nitrate 0.2 g L-1,Mn2+2.67 mmol L-1,pH=5.5,150 r min-1 shaking culture,70 mL culture solution in 250 mL flack,inoculation quantity 9 mycelium discs with diameter 8 mm and culture temperature 34?.After that,the ability of C.unicolor CB1 to degrade 5 kinds of dyes by extracellular enzymes from it were studied,and it was showed that the azo dyes could be more easily degraded than triphenylmethane dyes and Heterocyclic dyes by extracellular enzymes from C.unicolor CB1,the degradation rate of Congo red was 91.3%at day 4,and the degradation rate of Reactive Black was 95.9%at day 6,and the degradation rate of Reactive Red,Crystal violet and Neutral red was 89.4%,75.2%of and 72.3%of,respectively.4.As the application value of strain CB1 and their MnP was known,in order to comprehend the genetic traits,isozyme genes of mnp from C.unicolor CB1 were cloned by using Degenerate PCR,Genome Waking PCR and RACE technology.The full-length cdna and dna sequences of the three novel mnp genes were cloned and characterized from C unicolor,termed Cu-mnpl,Cu-mnp2,and Cu-mnp3.The full-length DNA sequences of Cu-mnpl,Cu-mnp2,and Cu-mnp3 was 4268 bp,3005 bp and 3001 bp,respectively.And the full-length cDNA sequence was 1092 bp,1429 bp and 1326 bp,respectively.Furthermore,Cu-mnpl genes consisted of a 1092 bp-ORF sequences interrupted by 15 introns,and Cu-mnp2 consisted of a 1128 bp-ORF sequences interrupted by 13 introns and Cu-mnp3 consisted of a 1083 bp-ORF interrupted by 5 introns.On the basis,nucleotide and amino acid of Cu-mnp1?3 were analyzed fully by bioinformatics?5.In order to understand the transcription mechanism of mnp isozymes genes,here,the expression pattern of Cu-mnp1?3 genes under different induce conditions were comprehensively studied.The results showed that the expression of Cu-mnp2 was higher than Cu-mnp1 and Cu-mnp3 under the normal culture conditions,while Cu-mnp1 and Cu-mnp3 could be highly induced by Mn2+and there was no significant effect on transcription level of Cu-mnp2 by adding to Mn2+in the culture.In this paper,this was the first time to analysis the effect of different dyes on transcription of Cu-mnp1?3 genes.It was showed that the expression patterns of Cu-mnp1?3 genes induced by Azo dyes(Reactive Black and Congo red)were similar.In the same,the expression levels of Cu-mnp1?3 improved in late culture stage.So it was speculated if the low concentration of dyes could promote the transcription of mnp genes and high concentration of dyes could inhibited transcription of mnp genes.In order to verify the speculation above,the effects of different concentration of Reactive Black on transcription of Cu-mnp1?3 genes were studied.The results showed that mnp genes could be induced highly by low concentrations of dyes.However,high concentrations of dyes inhibited expression of mnp genes.So we synthetically analyzed the effect of different types and concentrations of carbon and nitrogen sources on the transcription of Cu-mnp1?3 genes.The results revealed that the effect of different types and concentrations of carbon and nitrogen sources on expression of Cu-mnp1?3 genes were different.It was verified that MnP could be induced by multiple type and concentrations of carbons and nitrogen sources,and In other words,MnP gene families could be induced selectely by carbon and nitrogen sources,and it could be widely used.