| Phenoloxidase (EC.1.14.18.1, PO) is a key enzyme of insects, which can take part in normal developmental processes, such as cuticular tanning, scleration, wound healing, production of opsonins,encapsulation and nodule formation for defense against foreign pathogens, and so on. Therefor, to approach the effects of inhibitors including plant-derived compounds on the phenoloxidase (PO) of Spodoptera exigua (Hübner) and the immune functions of the enzyme, is helpful to the development of the"insect controller"harmonious with environment, a novel mode of action. Currently, many studies focused on this field in order to find effective inhibitors. Follows the previous works in the lab,, some properties of PO were investigated using microtitration in the present paper. The plant-derived compounds such as luteolin, podophyllotoxin, ursolic acid and oleanolic acid were selected to teste the inhibitory activities and kinetics on PO adopting the microtitrationassay method. Meanwhile, the bio-effects of these plant-derived compounds on the growth of Spodotera exigua larva with ingestion method were also investigated. Additionally, the preliminary study on the immune function of PO in the developmental processes of the larva were carried on in order to illustrat the importance of the enzyme.The contents and results were summarized as follows:1. The properties of PO partially purified were determined. The results indicated that the optimum temperature was 42℃, and the enzyme had a stable activity if the temperature reaction system is between 25-40℃; the optimum pH was 7.0 and the enzyme with a stable activity when the pH reaction system between 6.0-7.5. The effects of some metal ions on the PO activity were also studied and the results showed that Mg2+, Mn2+, Fe2+and Zn2+ cations could increase its activity,Cu2+ and Cu+ cations could activate the enzyme activity at the low concentration but inhibit its activity at the higher concentration. The results also indicated that the metal chelating agents such as Ethylene diamine tetraacetic Acid (EDTA) and Diethyl-dithio carbamic acid silver salt (DETC) could inhibit the enzyme activity; some organic solvents such as carbinol, ethanol, acetone and dimethylbenzene, had inhibitory effects on the PO activity. Benzene and Dimethyl sulfoxide (DMSO) could activate the enzyme activity at the low concentration but inhibit its activity at the relatively high concectrations.2. The inhibitory effects on the PO activity by the plant-derived compounds such as luteolin, podophyllotoxin, ursolic acid and oleanolic acid were determined and the possible mechanism of these inhibitors were also discussed in the dissertation. The results showed that all of the tested compounds could inhibit the enzyme activity and the inhibitor concentrations leading to a loss of 50% activity (IC50) were estimated to be 13.11 mmol·L-1, 0.2500 mmol·L-1, 0.3385 mmol·L-1 and 0.1701 mmol·L-1, respectively. All the tested compounds were reversible competitive inhibitors on PO and the Ki values were determined to be 0.2300 mmol·L-1, 0.0895 mmol·L-1, 0.2013 mmol·L-1, and 0.09709 mmol·L-1 respectively. On the basis of theoretical analysis, the inhibite mechanism was infered that the inhibition by luteolin was related to the structural homology between the inhibitor and the substrate,the inhibition by podophyllotoxin was likely to be related to its ability to remove oxidizing substance,the inhibition by ursolic acid maybe have something to do with its carboxyl group, and the inhibition by oleanolic acid may have some connection with its carboxyl group or its ability to change the protein conformation.3. Four plant-derived compounds, luteolin, podophyllotoxin, ursolic acid and oleanolic acid were selected to investigate the bio-effects on the growth of Spodotera exigua larva with ingestion method. The results showed that these compounds could posses its toxicity against little insect larva (≦3rd instar), and the LC50 value of the compounds against the tested larva were 2.53 g·L-1,1.84 g·L-1,6.80 g·L-1 and 6.04 g·L-1 at 11 d after treatment, respectively. In addition, these four compounds could obviously inhibit the growth and development of the larva(≧3rd instar), and the inhibition of podophyllotoxin was the highest inhibitor among the chemicals at 48 h after treatment, the inhibition rates of weight gain were 60.01% at the concentrations of 2.50 g·L-1, while the inhibition of ursolic acid was the lowest among the tested compounds and the results showed that the inhibition rates of weight gain were 62.58% at the concentrations of 16.0 g·L-1 under 48 h after treatment,. Theses compounds also had inhibitory effect on the pupation or eclosion against the insect larva and both the rate of pupation and eclosion was lower evidently than the corresponding data in the control groups, and was decreased combining with the increased concentrations of compounds. The toxicity for inhibiting the development of the tested insect were different. Luteolin was the highest toxicity and ursolic acid was the lowest botanical. The average pupal weight at concentration of 1.60 g·L-1 was 0.0647 g per pupa, the pupation and eclosion percentage were 36.67 % and 36.67% for luteolin and the latter showed that the inhibition of, at concentration of 16.0 g·L-1, the average pupal weight was 0.0755 g per pupa or the rate of pupation and eclosion was 53.33% and46.67 %, respectively.4. Relationship between phenoloxidase and the immune response was investigated to the insect, Spodotera exigua larva, after immune challenged by Escherichia coli (DH5αstrain). The results showed that, the PO activity increased 112% compared with treatment CK when E. coli incubated with hemolymph at 0℃; after the hemolymph was centrifuged to remove the hemocytes, the supernatant used as plasma was incubated with E. coli at 0℃, and its PO activity was as same as control; while the PO activity was raised accordingly with the increase of trypsin after the plasma incubated with trypsin solutions, which indicated that the plasma contain pro-phenoloxidase (proPO) The data above indicated that the recognition factor to E. coli may distributed on the hemocytes or other big molecules (such as fat body), not in the plasma. After the E. coli was injected to the body of larva for 2 h, some black nodules were formatted which indicated that the immune system of larvae responded to invaders soon after the infection at the same time the number of nodule increased as time prolonging and reached its apex at 10 h later. At the same time the PO activity changed obviously with the nodules formation. Within 4 h after the E. coli was injected to the larva body the activity of PO showed a sharp decline at beginning, then the downward trend became gently. It fell into the lowest at 13 h after treatment, and then began to increase slowly until PO activity returned to a normal condition at 25 h or so. The results indicated that the phenoloxdiase participated in the formation of nodules."...
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