The Determination Of The Melanin Synthesis Pathway Of Neurospora Crassa And The Screening And Cloning Of Related Genes

Natural sources of melanin is a kind of widely used biologicalpigment.The origin of melanin is different,result in melanin structure have bigger different,which have a close relationship by the melanin biosynthesis.As fungi research pattern N.crassa have the ability to produce melanin,study of N.crassa melanin synthesis pathway,find the key of the melanin synthesis genes,provide the foundation for building the melanin production engineering strains.This study from two ways the microbial synthesis of melanin?DHN way and L-DOPA way?,aimed to find the premise of bacterium which is used to synthetic melanin by rough vein bacterium,and synthesis of restraining factors,to determine the rough vein melanin synthesis way of N.crassa.The results showed that DHN approaches in the way the precursor of acetic acid as the substrate,N.crassa not be induced to produce melanin at normal culture conditions.To determine the rough vein bacterium melanin synthesis approach is not DHN way.L-DOPA way research results show that the tyrosine premised of melanin material synthesis which could be induced to produce melanin,the tyrosine content is 0.025g/50mL,culture temperature is 30??Shaking table number is 150rpm,the highest melanin production.At the same time,We further proved that the N.crassa produce melanin after tyrosinase oxidation tyrosine is a key step,by Kojic acid was used inhibitors of Key enzymes of L-DOPA way.With the increase of concentration of kojic acid in the process of N.crassa fermentation culture,result in the ability of production of melanin of N.crassa,when the concentration of kojic acid is 16mg/mL,the ability of production melanin is inhibited completely.In the infrared spectrum experiment of producing melanin N.crassa,we found that in1654.73cm^-1 there have a C=C that can produce a absorption peak,the absorption peak has a strong stretching and vibration.This functional groups is L-DOPA melanin feature groups.We identified the synthesis of melanin of N.crassais L-DOPA way through the above three aspects.The key enzymes of the synthesis of melanin is tyrosinase in L-DOPA way,we have used N.crassaDatabase to find that the relation genes associated with enzymatic synthesisisNCU09199?NCU11382?NCU08615 and NCU00776 in N.crassa,got the single gene deletion mutant strain of the four genes.For each of these four mutant strain fermentation to produce melanin.The results show thatthe lack NCU09199?NCU11382and NCU00776of gene has no effect on melanin production,at the same time deletion NCU08615 mutant strains also decreased significantly ability of produces melanin.So we think that the gene is key gene for the N.crassa produce melanin.According to NCU08615 gene sequence design primers,we have got the gene from WT 4200 of N.crassa strain,the size was about 2900bp,which was turned the gene and build the over-expression strainby testing of Western,expression protein is90kDa.Got higher expression of strain of tyrosinase.The study of the fermentation to producemelanin to tyrosinase,the ability of this strain of produce melanin 86%higher than wild typeWT 4200 of N.crassa strains.