Identification Of Mutant Genes In Caenorhabditis Elegans Mutant Conferring Resistance To Aurovertin D

Polyketides compound aurovertins are yello pigment secondary metabolites can be isolated and purified from Potato Dextrose Medium (PDB) fermentation broth of nematopathagenic fungus Pochonia chlamydosporia YMF1.00613. Aurovertin D displayed acute nematodetoxic activities towards root knot nematode Panagrellus redivivus and free living nematode Caenorhabditis elegans. Recent studies showed that aurovertin B could bind to β subunit of Adenosine Triphosphate synthetase (ATPase) to inhibit the synthesis of ATP. However, the mode of action of aurovertin D in nematode is unknown.30000C. elegans N2nematodes were treated with chemical mutagen ethyl methanesulfonate (EMS) to generate mutants which can resist to aurovertin D. The aim of this study is to identify and map the gene in mutagenised C. elegans responsible for resistance to aurovertin D. The priliminary study of the toxicity of aurovertin D to some pathogenic fungi was also included.Nematode mutant kyIs262was applied to identify the dominant mutation of the mutagenised C. elegans confers resistance to aurovertin D. Among the4mutants, one muntant (Ml) was characterized as dominant mutation after detecting the resistance of the progeny F1of mutants crossed with kyIs262worms. Nematode mutants MT464, MT465and CB4856with Single Nucleotide Polymorphisms (SNP) molecular markers were used to map the gene to a chromosome and internal region, and it was mapped to-2.97cM～-1.57cM in genetic map of chromosome Ⅲ, including0.93Megabase and149genes. Subsequently, the whole-genome resequencing of ml was performed and compared with reference genome sequence of wild-type N2.26variants were found in chromosome Ⅲ-2.97cM～-1.57cM of genetic map with8single-nucleotide variants,10insert and deletion variants,6structure variants and2copy number variants.3of them are variants in exons. PCR-method was applied to confirm the variants and only2are identified as mutagenesis-induced mutations,13are variations in the original transgenic strain,4are sequencing errors,7have not been confirmed becanse of the failure of PCR-amplify. In exon variants, there are2sequencing errors and1true mutagenesis-induced variant. The ture mutation was characterized to locate in atp-2. The change of one base G to A resulted in the changes in an amino acid varying from arginine to histidine. Then we injected the expression vector with atp-2gene and its promoter into C. elegans to express ATP-2. The N2worms injected with the plasmid containing the mutant atp-2gene displayed resistance to aurovertin D, indicating the relevance between the single-nucleotide variant in atp-2of C. elegans and the resistance to aurovertin D.This study successfully identified the resistance gene to aurovertin D in mutant C.elegans. The target of aurovertin D in C. elegans nematode has been investigated for the first time.The toxicity of fermentation broth of P. chlamydosporia YMF1.00613to6plant pathogenic fungi was primarily studied.