邻苯二甲酸酯暴露对凡纳滨对虾蜕壳生长的分子调控机理

Litopenaeus vannamei is one of main aquiculture in china. Phthalicacidesters is one of common organic pollutants which are enriched in aquatic organism. It belongs to endocrine disrupting chemicals and affects seriously aquiculture. So Litopenaeus vannamei is taken as experimental animal in our lab. By field survey and vivi experiments, after Litopenaeus vannamei is bred for a few days under the environment containing phthalicacidesters, the amount of phthalicacidesters enriched in Litopenaeus vannamei is determined. At the same time, the molting and growth of Litopenaeus vannamei are analysed. The effects of phthalicacidesters on the ecdysone and chitinase of Litopenaeus vannamei are investigated. The differences of mRNA expression, protein expression and activity of chitinase are studied. According to the experimental data, the dynamic relationship and pertinence of exoteric phthalic acid esters, internal ecdysone and chitinase are illuminated for Litopenaeus vannamei. The molecular response mechanism of chitinase of Litopenaeus vannamei to phthalic acid esters will be expatiated. The results will conduce to know the regulation mechanism of chitinase expression and the important role of chitinase in immunity defense of Litopenaeus vannamei, another will administer to explain the damage mechanism of phthalicacidesters for Litopenaeus vannamei aquiculture.

凡纳滨对虾在对虾养殖产业占很大比例，但目前病害现象严重，其中蜕壳紊 乱是病害的主因之一。对虾周期性蜕壳虽由内分泌激素调节，但也受外界因素干扰。邻苯二 甲酸酯（PAEs）是水体主要的内分泌干扰污染物之一，我们实验发现PAEs导致虾蜕壳变慢；而几丁质酶又与对虾的蜕壳生长密切相关。为阐明PAEs对对虾蜕壳生长的影响机制，我们首先调查PAEs在养殖场水体和虾体的富集情况，分析PAEs 污染与蜕壳激素含量和几丁质酶系活力的关系。在此基础上，进行活体暴露实验，测定不同PAEs 暴露下虾蜕壳生长状况、体内蜕壳激素及其受体，几丁质酶的变化（包含mRNA、蛋白及酶活力水平）；明确PAEs 与虾体蜕壳激素及几丁质酶系三者的动态关系及相关性，最终综合养殖场实地调查及实验室暴露，尝试揭示PAEs暴露对虾的蜕壳生长的影响及其分子机理，，为虾的健康养殖提供参考。

为了解水体PAEs 与凡纳滨对虾健康养殖的相关性。项目调查PAEs 在对虾养殖场水体的富集情况和对虾的生长状态，调查显示水体PAEs含量高于6ppb时，对虾生长处于亚健康或患病状态。在此基础上，为了进一步阐明PAEs 对对虾健康生长发育的影响机制，通过暴露实验研究邻苯二甲酸二甲酯（DMP）、邻苯二甲酸二乙酯（DEP）、邻苯二甲酸二辛酯（DOP）等PAEs物质及其可能的降解产物烷基酚等物质对凡纳滨对虾死亡率、蜕壳生长发育密切相关的几丁质酶系的影响，采用酶促反应动力学法测定几丁质酶系活力，以β-actin为内参，用western blot测定酶蛋白表达量、实时荧光定量PCR测定酶mRNA表达量。结果显示DMP、DEP、DOP、辛基酚、壬基酚、甲酚等有机物暴露均使对虾死亡率上升；对壳膜NAGase比活力影响基本起激活作用，但对内脏NAGase比活力影响不显著或起抑制作用；酶蛋白表达量没有明显变化规律；DMP对壳膜几丁质酶比活力影响不显著但对内脏几丁质酶活力起着激活作用；但DEP对内脏几丁质酶起着抑制作用。DMP暴露对壳膜NAGase mRNA表达量影响不显著，但随着暴露时间延长，壳膜几丁质酶 mRNA和内脏NAGase mRNA表达量显著下降，内脏几丁质酶 mRNA被激活。而DEP暴露对壳膜NAGase、内脏NAGase、壳膜几丁质酶的 mRNA表达量影响基本不显著，但随着暴露时间延长，内脏几丁质酶 mRNA表达量下降。实验结果说明PAEs对对虾毒害作用与对虾几丁质酶系活力和基因受损有一定相关性。