| Inonotus obliquus is a very valuable polyporaceae inonotus of medicinal fungi,which is mainly parasitizes on the bark or tree trunk of birch or other kind of trees,and its fruiting body presents irregular black tubercle.It is mainly distributed in the cold regions of high latitudes in the Northern Hemisphere,such as Poland,Finland,Russia,as well as Heilongjiang and Jilin of China,etc.,Inonotus obliquus has a variety of pharmacological activities,such as anti-cancer,anti-aging,enhance immunity and so on.Melanin biosynthesis is the important survival strategy for pathogenic fungi to resist environmental stress.Fungal melanins have been thought to be the products of DHN and DOPA pathway.However,biosynthesis of water-soluble melanin(WSM)by basidiomycetous fungus Inonotus obliquus cannot be inhibited by the pathway-specific inhibitors tricyclazole or arbutin.Previous work showed that physico-chemical properties of WSM by I.obliquus is very similar to the product of a gene encoding hybrid NRPSPKS from the fungus.Moreover,this nrps-pks formed a gene cluster with other 10 genes including those encoding protein kinase(cluster activation related),dipetidase(structure modification related)and haemolysin co-regulated protein(transmenbrane transportation related).In order to study the relevant functions of NPM(Nonribosomal-peptide Polyketide Melanin)gene cluster of Inonotus obliquus,in this paper,the following research will be carried out on the basis of early laboratory research:1.Based on the molecular genetic manipulation system of Aspergillus nidulans,the NPM gene cluster expressing strain in Aspergillus nidulans was constructed by homologous recombination and fusion of PCR technique.2.Based on the expression system of Saccharomyces cerevisiae heterologous,and take the plasmid recombination technique as the method to construct the piecewise expressing strain of Saccharomyces cerevisiae of nrps-pks gene.3.Based on the expression system of NICE protein of Lactococcus lactis,and take the plasmid recombination technique as the method to construct the expressing strain of HCP gene of Lactococcus lactis.4.Based on the strain transferred to nrps-pks gene and NPM cluster at the w A site of Aspergillus nidulans,and use the four different amino acids as substrates,to study the phenotypic changes.The experimental results showed that the NPM gene cluster of Inonotus obliquus was successfully and completely transferred into wA site of Aspergillus nidulans through 10 times of piecewise transformation.And the nrps-GST-pXW55 carrier and nrps-pksGST-pXW55 carrier constructed by plasmid recombination technique were successfully expressed in Saccharomyces cerevisiae.The HCP-pNZ8148 constructed by plasmid recombination technique was successfully expressed in Lactococcus lactis.After the induction of four amino acids as substrates,the phenotypic changes appeared in different degrees in the culture media,especially when Phenylalanine and Tryptophan and Isoleucine were used as substrates.Our results show that the NRPS-PKS incorporates most preferably the Ile,Phe and Trp.This indicates that this NRPS-PKS hybrid enzyme possesses substrate specificity.Although this study was based on the construction of NPM gene cluster of Inonotus obliquus and the relevant gene heterologous expression strain,which will provide the necessary materials for the further study of nrps-pks expression product structure unit,chemical structure and the functional studies of genes closely related to nrps-pks transcriptional activation,structural modification of expression products and transmembrane transport in NPM gene cluster,and it will lay a foundation for the discovery of a new pathway of melanin synthesis. |
PDF Full Text Request