ABSTRACT
In order to isolate the genes expressed specifically and abundantly in the mature fruit body of Lentinula edodes, the cDNAs derived from the gill of the fruit body were compared with the cDNAs from the mycelia by differential screening. Consequently, six clones were identified as fruit-body-specific genes (fbg03, 08, 13, 14, 16, and 21). The deduced amino acid sequence of fbg14 (Le.cypfb) had significant homology with the cytochrome P450 protein. The transcriptional level of fbg16, which showed 29.9% identity with the riboflavin aldehyde-forming enzyme of Agaricus bisporus, was highest among all of the fbg clones. This result indicates that the promoter region of fbg16 may become a powerful candidate for the expression signal of the vector for the gene manipulation in the mature fruit body.
Subject(s)
Gene Expression Regulation, Fungal/physiology , Genes, Fungal/genetics , Lentinula/genetics , Amino Acid Sequence , Blotting, Northern , Cloning, Molecular , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , DNA, Fungal/biosynthesis , DNA, Fungal/genetics , Gene Library , Molecular Sequence Data , Transformation, GeneticABSTRACT
We cloned a gene for the iron sulfur protein (Ip) subunit from an edible mushroom, Lentinula edodes, and introduced a point mutation that confers carboxin resistance into it. The mutant gene successfully transformed L. edodes with high efficiency (9 transformants/2.5 microg vector DNA). Restriction enzyme-mediated integration (REMI) increased the transformation efficiency by about two-fold.
Subject(s)
Genetic Markers/genetics , Shiitake Mushrooms/genetics , Transformation, Genetic/genetics , Blotting, Southern , Carboxin/pharmacology , Cloning, Molecular , DNA Restriction Enzymes/metabolism , Drug Resistance/genetics , Genetic Vectors , Iron-Sulfur Proteins/genetics , Point Mutation , Protein SubunitsABSTRACT
We identified and analyzed the dffA gene from Aspergillus oryzae which encodes L-ornithine N5-oxygenase involved in the biosynthesis of deferriferrichrysin, a type of siderophore which is a low-molecular-weight iron chelating compound. From among more than 20,000 clones in an A. oryzae EST (expressed sequence tag) library, we found only one clone encoding a protein that exhibited homology to theUstilago maydis sid1 protein (Sid1) and Pseudomonas aeruginosa pvdA protein (PvdA), both known as the only examples of L-ornithine N5-oxygenase. The complete gene sequence shows that the dffA gene includes a 1575-bp open reading frame (ORF), one 66-bp intorn, which is a typical intorn length inA. oryzae, and encodes 502 amino acids with putative FAD-binding, NADP-binding, and 'FATGY' motifs, which are conserved inN-hydroxylating enzymes. As well as that of the U. maydis sid1 gene,dffA gene expression was induced under iron-limited conditions, and the promoter region has several GATA-type transcription regulator binding motifs. When the dffA gene was expressed under the control of the a-amylase promoter in A. oryzae, transformants revealed inducible high L-ornithine N5-oxygenase activities. In addition, a dffA gene disruptant showed no deferriferrichrysin production even under iron-limited conditions. These results clearly suggest that the dffA gene is indispensable for deferriferrichrysin biosynthesis in A. oryzae.
