Cloning And Functional Analysis Of The Melanin Biosynthesis Gene 4HNR In Setosphaeria Turcia

In order to identify the melanin biosynthetic pathway and the relationship between melanin and the pathogenicity of Setosphaeria turcica,1,3,6,8-tetra-HN reductase(4HNR) which belong to the melanin biosynthetic pathway were cloned and the functional research were done by bioinformatics and mutants analysis,meanwhile analysis of gene expression was completed.It was conclusion that 4HNR gene might play an important role in melanin biosynthetic pathway and pathogenicity ofS.turcica.Genome DNA and cDNA homologous fragments of the 4HNR gene were obtained by polymerase chain reaction(PCR)amplification with specific primers which sets designed on the basis of the conserved nucleotide regions of 4HNR conservative domain from others fungis known.The completed DNA sequence was been accessed through Genomic walking. Both sequences of DNA and cDNA of 4HNR are 807 bp and there is no intron in this sequence through alignment.It has a completely open reading frame composed of 268 codons,the molecular weight is about 28.484 kD,and pI 6.52.It is includes 118 hydrophobic residues and 150 hydrophilic residues,furthermore,a complete 3-ketoacyl-(acyl-carrier-protein)reductase domain has been found.This sequence was been submitted on the GenBank,and the GenBank accession of nucleotide is EU494943 and the protein is ACA52027.The deduced amino acid sequence of the 4HNR showed high similarity to the protein sequence with tetrahydroxynaphthalene reductase in Cochliobolus heterostrophus and Neurospora crassa,96%and 78%respectively.The specific probe of 4HNR was be preparation,and to apply for Southern blotting.Double positive of stripe were been found in the production of enzyme digest with single enzyme site and single positive of stripe was been found in the production of enzyme digest with none enzyme site,so this gene only has single copy in genomic.A 2 285 bp for the flanking sequence of 5' and 301 bp of 3' have been obtained through Genimic walking,and it has promoter structure within the framework of the 1 000 bp before the start codon through the software analysis of Softberry and NNPP.A TATA box was been found in -34 and a CAAT box in -70,etc.After,the vector of eukaryotic expression for target gene was been constructed and expressed in host yeast strain GS 118 through electroporation and PEG1000,the 4HNR protein was been secreted into the supernatant of yeast strain.The vector of double-cross homologous recombination of 4HNR was been constructed,and the protoplasts of S. turcica were been transformated through PEG4000.31 transformates were obtained,and a transformate showed gray color.It is speculated that the transformate was mutant through PCR verification.The research was completed in the genes cloning and expression,and the mutant of gene deletion,which layed foundation for making new fungicides.