Cloning Of Cytochrome B₅ And Cytochrome B₅ Reductase Genes And Functional Research About MATE Transporters In Penicillium Digitatum

Penicillium digitatum is a typical necrotrophic pathogen that often infect citrus fruits,lead to green mold.P.digitatum is responsible for up to 90%of the total losses during postharvest packing,storage,transportation,and marketing.Imidazoles bactericide can effectively control green mold,but the long-term use of bactericide lead to strain resistance.So it is necessary to explore other mechanisms to control the green mold.The objective of this study is to investigate the relationship between cytochrome b5?Cyt b5?and cytochrome b5 reductase?Cyt b5r?with the cytochrome P450 CYP51A on the function of electron transfer and pesticide resistance mechanism of MATE family transporters in the P.digitatum,providing theoretical basis for development of new type bactericide.The main results are as follows:1.Thro?gh analying the transcriptome results,citrus fruits P.digitatum as the research material,the whole sequence of cyt b5 and cyt b5r genes were screened and cloned by PCR,named Pdcyt b5 and Pdcyt b5r respectively.Gene sequences and protein sequences were analyzed by bioinformatics softwares,and found that Pdcyt b5r contained a RXY?T/S?XX?S/N?domain,corresponding position R301 to S307.Recombinant plasmid pET-cyt b5 and pET-cyt b5r were expressed successfully in E.coli.2.Recombinant plasmid ppbrA?pPlC-Pdcyp51A-cyt b5-cyt b5r?was constructed successfully.This recombinant plasmid was transformed into Pichia pastoris by electroporation and CYP51A was coexpressed with Cyt b5-Cyt b5r.Real-time PCR technique was used to analyze the expression level of these three genes.Real-time PCR analysis results showed that the expression level of cyp51A coexpressed with cyt b5-cyt b5r in P.pastoris was 54-97%higher than that when it was expressed alone over an extended period?48-72 h?.It is concluded that Cyt b5-Cyt b5r complex was capable of transferring electrons to PdCYP51A,and they can enhance the expression level of cyp51A.As target enzyme of antifungal agents,the high level expression of CYP51A is closely related to the fungus resistance.It is of vital significance in the study of fungal resistance mechanism and the designation of the new dr?g.To our knowledge,this is the first report to explore the function of cyp51A gene by co-expressing with cyt bs-cyt b5r complex in P.digitatum,3.We have analysised the total RNA of P.digitatum resistant strains?HS-F6?and the sensitive strain?HS-E3?thro ? gh transcriptome sequencing.The results indicate that the expression level of three Multidr?g efflux transporter coding genes were higher in HS-F6 than in HS-E3.Homologous sequence alignment?NCBI Blast?presumpted that they belong to MATE?Multidr?g And Toxic Compound Extrusion?family.Full length of the three genes were obtained by PCR,as Pdmatel?PDIG₄2350m.01?933 bp,Pdmate2?PDIG₃5850m.01?862 bp,and Pdmate3?PDIG₂5390m.01?1477 bp.The homology of three MATE proteins were 23.73%to 23.73%.Real-time PCR analysis results showed that the expression level of Pdmates in HS-F6 were upregulated after induction by Prochloraz,especially Pdmate2 and Pdmate3;while expression level of Pdmates in HS-E3 were downregulated.After induction by imazalil,the expression level of Pdmates in HS-F6 were upregulated,especially Pdmatel;while expression level of Pdmates in HS-E3 were downregulated.These results are consistent with the analysis of transcriptome.Three Pdmates genes were all pregulated after induction,but responsing genes were different.It might means that the three genes have different dr?g sensitivity and specificity.Excess expression of PdMATEs were likely associated with dr?g resistance of HS-F6.4.The best way to understand the roles of the MATE transporters in each organism is via the construction of specific deletion mutants and analysis of the mutants.We constructed two deletion mutants APdmate2 use agrobacterium mediated reforming process,and this will lay the foundation for the further research on MATE family transporters about sporulation rate,virulence and dr?g resistance mechanism in P.digitatum.