Studies on the ATPase of the Diamondback Moth Plutella Xylostella(L.)(Lepidoptera Plutellidae)and Its Inhibiors
|School||Hunan Agricultural University|
|Keywords||Plutellaxylostella ATPase inhibitor gene expression|
The adenosine triphosphatase, referred to as ATP synthase (ATPase), was widely found in different organisms, such as microorganisms plants and animals. ATPase could catalyze ATP hydrolyzing to ADP and Pi. The hydrolysis released energy which was used to control the different ion concentration of inside and outside neurilemma, The ATPase could also catalyze ADP and Pi synthetizing to ATP. It was one of the most important enzymes in insect, and involved in major physiological functions of the insects, such as controlling conduction of the nerve system, providing energy for absorption of amino acids. The research on enzyme of ATPase from insects was important to pest control. In this paper we have done research on the properties of the ATPase and the effects of some effectors on its activity from diamondback moth P. Xylostella(L.) by conventional methods in the laboratory. The results was summarized as follow:1. The activity of ATPase from P. Xylostella(L) in different stages and instars was different. The activity of total ATPase was gradual increased with the larval instar from second to forth. And the enzyme activity of the4th instar larva was the highest, which was17.236U/mgprot. However it decreased in the pupal period, while two-day pupae was the lowest, with a degree of8.554U/mgprot.2. The properties of ATPase showed that the optimum value pH for determination of ATPase activity was7.4and the optimum temperature was37℃.The Michaelis constant (Km) of ATPase hydrolyzing ATP was0.151mmol/L and the maximum velocities(Vmax) hydrolyzing ATP was0.676μmol/(mg-h).3. The effects of some metal ions on the total ATPase activity was studied. The results showed that Mg2+、Mn2+、Fe2+could inhibit the ATPase activity. When the concentration was ranged from1.25X10-4～20×10-4mol/L, the inhibition rate was fluctuated between5.67%-77.58%.While K+increased the activity at the low concentration (1.25×10-4mol/L), the enzyme was in the strongest activation which was18.55%.4. The effects of some organic solvents on the total ATPase activity was studied. The results showed that ethanol, acetone, dimethylbenzene and ethyl acetate had inhibitory effects on the enzyme activity. When the organic solvent volume was400μL, the inhibition rate were1.83%,1.62%,6.36%and4.07%respectively. When the organic solvent volume was2000μL, the rate were separately85.06%,51.29%,47.74%and100%. The inhibition of enzyme activity was significantly increased with the increasing of the organic solvent volume.5. By the studying of nine insecticides on the inhibition of ATPase activity, the results showed that pyrethrin (46.99%) was the highest under the same concentration and the second was organophosphorus(profenofos and chlorpyrifos) which was46.01%and45.46%respectively. The following was the carbamate (fenobucarb and methomyl) with rates of44.62%and34.46%seperately. The percentage of inhibition of.ATPase activity by remaining insecticides including imidaclprid, thiamethoxam, dinotefuran and spinosad was all less than21%. The inhibition of ATPase activity by seven insecticides was affected with the concentration changed.6. The differences of P. Xylostella(L.)V-ATPase β subunit gene expression were determined with real time PCR technology. The results showed that the gene expression was different in different developmental stages of P. Xylostella and the expression of adult stage was the highest and the pupae was lowest, which was2.29and0.68folds respectively compared to the1st instar larvae.