Researching of gene region responsible for ribotoxin production in Aspergillus species isolated from animal feed in the market

The aim of study is researching gene region responsible for ribotoxin production in Aspergillus species which belongs to the genus Aspergillus isolated from animal feed in the market. When the studies are examined, among the ribotoxin producing fungi, the excess of species belonging Aspergillus genus is remarkable. Ribotoxins are a family of ribosome inactivator proteins which have a fairly high specific activity against the phosphodiester bond of the sarcin/ricin loop universally preserved in 28S rRNA, leading to protein synthesis inhibition and cell death by apoptosis. In our study, 16 animal feed taken from places which selling cattle and sheep feed in Aegean Region were used. All of the fungi isolated from these feed samples were identified by phenotypic methods, and the identification of the 27 isolates belonging to Aspergillus genus was confirmed by genotypic methods. For the genotypic diagnosis, the polymerase chain reaction (PCR) method was performed using the universal primers ITS1 and ITS4 which spesific for the ITS gene region conserved in the fungus. The resulting PCR products were submitted for sequence analysis. Sequence results from the analysis were evaluated using Finch TV (Blast) and ApE program. The species identification was made by examining the similarities of the nucleotide sequences in the Gene Bank's https://blast.ncbi.nlm.nih.gov/Blast.cgi (NCBI) website, which is open to the use of researchers. As a result of these researchs, 13 different Aspergillus species were detected in 27 Aspergillus isolates. In order to research the presence of the ribotoxin gene region in all genotypically identified Aspergillus strains, PCR was performed with KQ1-KQ2 primers that were specific for the ribotoxin gene region in the fungi. Sequence analysis of all the obtained products was carried out and the results were examined on the NCBI page. As a result, the presence of mitogillin family cytotoxin clavin / c-sarcin gene region in an Aspergillus clavatus species and the presence of mitogillin family Aspf 1 ribotoxin gene region in an Aspergillus fumigatus species from 27 Aspergillus isolates. In addition, it was determined that 95% sequence homology with the partial mRNA of Aspergillus aculeatus hypothetical protein was shown by the sequence analysis of the PCR product obtained from the Aspergillus aculeatus strain after PCR with the primers specific to the ribotoxin gene region. Considering that new ribotoxin varieties are being discovered every passing day, future studies suggest that this protein region may also be linked to ribotoxins.