Cloning And Sequence Analysis Of Six Antioxidant Enzymes Of Ditylenchus Destructor And Effects Of Low-dose Nematicides On Their Expression

Potato rot stem nQmatode(Ditylenchus destructor) is one of important pathogens of sweet potato in China, and the enormous yield losses to agriculture is very great annually. After the nematicide application, rudimental nematicide exists mainly in the form of low-dose in soil, low doses of nematicides may produce reactive oxygen spcies, thus contributing to harmful organisms, however, the organisms will geneate antioxidant enzyme to against those damages.In this study, six antioxide genes including extracellular CuZn superoxide dismutase(EC-CuZnSOD), cytosolic CuZn superoxide dismutase(CT-CuZn SOD), manganese superoxide dismutase(MnSOD), Peroxiredoxins(Prx), Glutathione Peoxidases(GPx) and Catalase(CAT) were cloned from D. destructor using RT-PCR and RACE, the mRNA expression of those antioxide genes were detected at24h after soaking in low dose(10、1、0.1、0.01、0.001and0.0001μg/ml)nematicides of emanectin benzoate, aldicarb and profenofor using Real-time PCR after24h.The sequence analysis results showed that:The full-length cDNA of Dd-EC-CuZnSOD-1, Dd-CT-CuZnSOD-1and Dd-MnSOD-1were848bp,680bp and1006bp, respectively. The ORF encodes217,159and221amino acids, respecitvely. The GenBank accession No. are KC460330, KC460329and JQ609354. The putative proteins of Dd-EC-CuZnSOD-1contains a19aa signal peptide at the N-terminal and two transmembrane domains but absented in other putative proteins. The genomic DNA of those genes are1673bp,1597bp and2090bp, respectively, the intron analysis showed that the Dd-EC-CuZnSOD-l and Dd-MnSOD-1included6extons and5introns, meanwhile, the CT-CuZn SOD-1included4extons and3introns.The full-length cDNA of Dd-Prx-1, Dd-GPx-1, and Dd-CAT-1were784bp,950bp and1753bp, respectively. The ORF encodes196,226and509amino acids, respectively. The GenBank accession were JQ609353, JQ609355and JQ609356. The signal peptide at the N-terminal were no found in the three putative proteins, but the Dd-Prx-1contains a transmembrane domains. The genomic DNA of those genes are2139bp,2706bp and5048bp, respectively, the intron analysis showed that the Dd-Prx-1, Dd-GPx-1, and Dd-CAT-1included5,3and15introns, and it contain6,4 and16extons respectively.The mRNA expression of D. destructor treated by low dose of(l0、1、0.1、0.01、0.001and0.0001μg/ml) nematicides of emanectin benzoate, aldicarb and profenofos were monitored by real-time PCR after24h. The results showed that:(1) The emamectin benzoate has more effect on GPx gene expression, the GPx gene upregulation expression of2.2times on the concentration of0.01μg/ml, when dealing with concentration of0.01μg/ml, EC-CuZn SOD gene expression raised1.2times, whereas when the concentrations is10μg g/ml, EC-CuZn SOD gene expression decline1times, when the concentration is0.01μg g/ml, CT-CuZn SOD gene expression has raised1.2times, and it almost no effect on the expression of Prx.(2) The aldicarb has more effect on EC-CuZn SOD and GPx, when the concentration is0.01μg/ml, EC-CuZn SOD gene upregulation expression2.07times, when dealing with concentrations of0.0001μg/ml, GPx expression increased2.3times, the aldicarb has less effect on Prx, by1.36times when conce-ntrations of0.0001μg/ml, it has no impact on CT-CuZn SOD.(3) The profenofos on EC-CuZn SOD, CT-CuZn SOD, GPx and Prx genes were upward in1.6to1.9times, when the concentration is1μg/ml, expression of EC-CuZn SOD increased by1.6times, when the concentration is1μg/ml, expression of CT-CuZn SOD increased by1.7times, when the concentration is1μg/ml, expression of Prx upregulation1.62times, when dealing with lower concentrations (0.0001,0.001,0.01,0.1and1μg/ml), GPx gene were increased between1.29tol.9times.The results presumed that emamectin benzoate which residues in soil has less effects on D. destructor, profenofos has more effects on D. destructor.