【作者】 谢桂英；

【导师】 罗万春；

【作者基本信息】 山东农业大学 ， 农药学， 2003， 硕士

【摘要】 当今，农药的发展正面临一个关键时期，发展对环境友好害虫控制剂的观念越来越深入人心，这个领域也越来越成为世界各国科学技术争相抢占的一个制高点。酚氧化酶（phenoloxidase，简称PO，对哺乳动物称为酪氨酸酶）广泛存在于生物体内，它是昆虫体内的一种重要酶类，在昆虫变态发育和免疫系统中起着重要的作用。酚氧化酶抑制剂理论可以为发展新型的害虫控制剂提供重要线索。本文通过室内实验，主要研究了曲酸等10种天然源化合物对不同龄期小菜蛾的生物活性，选出了几种对小菜蛾有活性的化合物；测定了它们对小菜蛾酚氧化酶的抑制活性和抑制类型；并对其生物活性与其对酚氧化酶抑制程度的关系进行了初步探讨；初步研究了小菜蛾酚氧化酶的基本性质；试图从酚氧化酶抑制剂角度为寻找新型害虫控制剂开辟一个崭新的领域。通过研究，首次报道了曲酸等10种天然源化合物对小菜蛾多个虫态的生物活性；首次报道了其中8种化合物对小菜蛾酚氧化酶的抑制程度和抑制类型；首次对小菜蛾体内酚氧化酶作了初步纯化并报道了其基本酶学性质。基本内容概要如下：1.供试的10种化合物对小菜蛾都表现出一定的生物活性。其中，触杀活性（点滴法对3龄）以对羟基苯甲酸活性最高，其次为2-羟基-4-甲氧基苯甲醛；其它化合物活性较低；点滴法处理4龄初幼虫，表现出曲酸、槲皮素显著影响最终成虫数；浸渍法处理小菜蛾2龄幼虫，以异香草酸活性最高；浸渍3龄幼虫则以槲皮素、对羟基苯甲酸的活性最高；对羟基苯甲酸、槲皮素对蛹羽化率的影响最大；槲皮素能明显影响1～2天卵的孵化，其它化合物的影响都不大；在浸叶饲喂法测定中发现槲皮素对小菜蛾的生长发育影响最甚。2.供试10种化合物除芦丁外，都能抑制小菜蛾酚氧化酶活性，其中以曲酸、槲皮素的抑制作用最强，其I50分别为66.54和94.99 μg/mL，其它化合物按由强到弱的顺序排列依次为：对羟基苯甲酸（157.26μg/mL）、间羟基苯甲酸（201.61μg/mL）、香草酸（268.22μg/mL）、5-甲氧基水杨酸<WP=7>（284.63μg/mL）、异香草酸（299.64μg/mL）、2，4-二甲氧基苯甲酸（309.99μg/mL）和2-羟基-4-甲氧基苯甲醛（385.30μg/mL）。3.供试10种化合物除芦丁、香草酸外，都是小菜蛾酚氧化酶的典型竞争性抑制剂，这些化合物与酚氧化酶的底物（如多巴、邻苯二酚、焦性没食子酸）结构相似，因而与底物竞争酶的结合部位，从而抑制酚氧化酶的活性。4.所试10种化合物对小菜蛾的生物活性（这里主要指致死活性）与其对酚氧化酶抑制活性之间没有很好的相关性。如曲酸对酚氧化酶的抑制活性最强而生物活性并不高，甚至很低；而对羟基苯甲酸、槲皮素对酚氧化酶的抑制活性不是最强而表现出生物活性很高的现象。5.研究报道了小菜蛾体内酚氧化酶的初步纯化及其基本酶学特性。该酶在pH6.0-7.0范围内有较高的稳定性；其最适pH 值为6.0；最适反应温度为40oC；在40 oC以下有较高的热稳定性，高于40oC则迅速失活。本文还对昆虫酚氧化酶的研究现状及与害虫控制剂的关系、天然产物用做害虫控制剂的前景以及关于寻找新型害虫控制剂的途径--生物合理设计的思考等问题进行了讨论更多还原

【Abstract】 Phenoloxidase (PO) had been shown to be present in organism and it is a key enzyme in the development of insect. As soon as the higer requirement to the environment people focus on the field where they want to find new style pesticides. As time flies the conception of Integrated Pest Mangement (IPM) had been changed to Integrated Agro-ecosystem Management(IAM), that is people do not want to kill all pests but to control the number of them to reduce the lose of agricultural output. In this study, some natural compounds like kojic acid were employed to test if they have the biological activity to diamondback moth Plutella xylostella and to testify if they can inhibit phenoloxidase from the tested insect. In this page,the relationship between biological activity and inhibitory degree of phenoloxidase was also discussed.Several methods were usued as bioassay in the test and the results showed us the contact toxicity or ingestion. In general those compounds did not have high biological activity for the highest death rate was lower than 50%. By topical application on 3rd larva p-hydroxybenzoic acid was found out to have higher biological activity than others but as for 4th larva kojic acid and quercetin can obviously reduce the number of adults appearing. When 2nd larva were dipped in the solution of the compounds, it was found that isoranillic acid was the most effective compound to kill the larva. Also it was p-hydroxybenzoic acid and quercetin that can affect emergence percentage markedly. All compounds except quercetin can hardly prevent eggs from hatching into larvae. By dipping leaves it was found that quercetin can affect growth development of diamondback moth evidently. On the contrary, some<WP=9>larva after treated were stimulated to add its weight.The results of those compounds’ inhibitory degree to phenoloxidase from diamondback moth indicated that all compounds except rutin can inhibit phenoloxidase. The sequence ordered by I50 was showed as below:kojic acid <quercetin< p-hydroxybenzoic < m-thdroxybenzoic acid < vanillic acid<5-methoxysalicylic acid <isovanillic acid <2,4-dimethoxybenzoic acid <2-hydroxy-4-methoxy-benzaldehyde <rutin. That means kojic acid showed the most activity to the phenoloxidase from the tested insect in this paper.The style of the inhibition on phenoloxidase from diamondback moth was also studied. The results showed that those compounds(execept rutin and vanillic acid) were competitive inhibitors. The scientific explain may be that all these compounds have a same part structure with the substrates which can be catalysised by phenoloxidase. The relationship between biological activity and inhibitory extent to phenoloxidase was also discussed in this paper. It was inferred that some compounds’ biological activity was not directly relatived with inhibitory activity of the phenoloxidase.The phenoloxidase from diamondback moth was purified and tested its characters. After phenoloxidase was purified by (NH4)2SO4 and SephadexG-100 gel filtration the higher activity enzyme was achieved. The enzyme characters of the phenoloxidase indicated that it can catalysis tyrosine, catechol and pyrogallol etc,and catechol suited to be substrate most in this paper. The optimum pH and temperature for the phenoloxidase was 6.0 and 40℃ and the activity of PO will be decreased when the temperature were above 40℃更多还原