The Regulatory Genes In Pks-6 Gene Cluster In Filamentous Fungus Neurospora Crassa

Neurospora crassa is a model fungus for the study of microbial genetics and molecular biology.In addition,it's the only filamentous fungus that possesses genomic sequences,functional genomic tools,and genome-wide knockout mutant libraries.What's more,N.crassa is a classic model organism as well as a commonly used strain for genetic research in the laboratory.There are six polyketide synthase genes(pks)in its genome,which are pks-1?pks-6.According to the prediction of the PFAM protein family database,the pks-6 gene cluster consists of 14 genes,NCU02918?NCU02931.However,anti SMASH predicts that the pks-6 gene cluster consists of 14 genes,NCU02913?NCU02926.The predictions of these two websites show that the composition of the pks-6 gene cluster is different.Therefore,the purpose of this experiment is to study which genes are composed of the pks-6 gene cluster and the relationship between part of the regulatory genes.Specifically,the wild-type Neurospora crassa 87-3 and deletion mutant strains(?NCU02917,?NCU02919,?NCU02921,?NCU02931)were cultivated under normal conditions and stress conditions(toothpick line,Drosophila melanogaster,Sinella curvisata),and then the differences about secondary metabolites and genes expression can be analyzed.According to the results of TLC,HPLC and QPCR,the boundary of the pks-6 gene cluster may exist within the span of two predictions,NCU02913?NCU02931.The results also demonstrated the relationship between partially regulated genes,such as low expression of pks-6 after the deletion of NCU02919;when ?NCU02919 and ?NCU02921 were devoured by Sinella curvisata,the expression of NCU02917 was higher;and when ?NCU02931 was devoured by Drosophila larvae and Sinella curvisata,the genes NCU02917,NCU02919,and NCU02921 all showed low expression.In addition,more data and conclusions are presented in this paper.It is worth noting that NCU02925 is a gene on the pks-6 gene cluster and it is also named fbm-1,because related studies have shown that this gene not only encodes FAD-binding/dehydrogenase/monooxygenase,but also participates in the maturation of fruiting bodies.It is found that the ascosporas can normally mature after knockout pks-6.That is to say that although they are in the same gene cluster,NCU02925(fbm-1)may be able to work independently of pks-6(NCU02918)during maturation of the ascosporas.So there are so many problems need further study,for example when the fbm-1 gene is expressed,and how the gene participates in the formation of the fruiting body.In this experiment,NCU02925(fbm-1)and green fluorescent protein gene(GFP)were firstly combined and inserted into the vector pqa-5Myc-6His to form a recombinant plasmid,which was transformed into ?NCU02925 strain by electrotransformation.Then according to observation by microscopy,the expression of fbm-1 can be analyzed by the existence of green fluorescence.As a result,it showed that fbm-1 was not expressed in young mycelia;there was a certain amount of expression during the maturation of the mycelium,and more expression was concentrated in the conidia area;high expression during asperity spore maturation especially at two symmetrical positions in the ascospores.In short,the results of this experiment provide further explanation for the function of the NCU02925(fbm-1)to participate in fruiting body formation.In a word,the result of this paper mainly proves that the limits of the pks-6 gene cluster may exist between NCU02913-NCU02931,and explains the relationship between partially regulatory genes.In addition to this,it also gives more specific explanations of the function of NCU02925.