Cloning And Functional Analysis Of The Melanin Biosynthesis Gene StPKS In Setosphaeria Turcica

In order to identify the melanin biosynthetic pathway, the StPKS gene was cloned and the function was researched by bioinformatics and mutants analysis. It was concluded that StPKS gene might play an important role in melanin biosynthetic pathway and pathogenicity of Setosphaeria turcica.The DNA sequence was obtained through Genome walking. Genome DNA and cDNA full sequence fragments of the StPKS gene were obtained by polymerase chain reaction (PCR) amplification with specific primers. The sequence of DNA was 6,527 bp and had one intron. It had a completely open reading frame composed of 2158 animo acid, the molecular weight was about 232.6663 kD, and pI6.00. It is included 955 hydrophobic residues and 1203 hydrophilic residues, furthermore, 5 complete motifs which are N-terminus of the protein, KS motif with the active site cysteine, Acyl transferase motif with the active site serine, Acyl carrier protein motif with the active site serine, TE motif with the active site serine had been found. The deduced amino acid sequence of the StPKS showed high similarity to the protein sequence with polyketide synthase in Bipolaris oryzae, Cochliobolus heterostrophus, Pyrenophora tritici-repentis and Phaeosphaeria nodorum, 91%, 89%, 84% and 79% respectively. The specific probe of StPKS was prepared, and to apply for Southern blotting. Single positive of stripe was found in the production of enzyme digest with none enzyme site, so this gene only has single copy in Genome. A 277 bp for the flanking sequence of 5′and 644 bp of 3′had been obtained through Genime walking, and it had promoter structure within the framework of the 170 bp before the start codon through the software analysis of Softberry and NNPP. A TATA box was found in -17, and so on. After vector of double-cross homologous recombination of StPKS was been constructed, and the protoplasts of S. turcica was transformated through PEG4000. 50 transformates were obtained, and 4 transformates showed gray color and non-pathogenicity. It is speculated that the transformate were mutant through PCR verification. The research was completed in the genes cloning and the mutant of gene deletion, which layed foundation for making new fungicides.