小峰熊蜂的乙酰胆碱酯酶时空表达特性研究
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摘要
熊蜂是濒危、高寒植物和设施农业的主要授粉昆虫，农药的施用被认为是全世界熊蜂数量和种类下降的主要原因之一。以乙酰胆碱酯酶(acetylcholinesterase, AChE)为靶标的有机磷和氨基甲酸酯类杀虫剂是我国目前应用最广泛的杀虫剂。为了合理保护和利用熊蜂，本文以我国分布最广泛、最具有商业发展前景的小峰熊蜂为研究对象，利用酶活测定法和荧光定量PCR法研究了小峰熊蜂AChE的时空表达特性。另外，通过AChE离体抑制法和生物测定法测定了有机磷和氨基甲酸酯类杀虫剂对小峰熊蜂AChE的抑制作用和毒力。全文主要结果如下：
1、利用正交试验方法优化得到了小峰熊蜂AChE最佳测定条件。结果表明：酶浓度0.25g蛋白质/L，底物浓度0.8mmol/L，pH值7.5，温度40℃，反应时间5min为小峰熊蜂AChE的最佳测定条件。这5个因素对小峰熊蜂头部AChE活性的测定均有极显著的影响（P≤0.01），影响大小依次为酶浓度、pH、温度、底物浓度和反应时间。
2、对小峰熊蜂AChE活性分布的研究及ace2基因相对表达量的分析明确了小峰熊蜂AChE的空间表达特性。组织分布结果表明，小峰熊蜂头部AChE活性（63.55%）显著高于胸部（18.44%）和腹部（18.01%）（P≤0.05）。进一步对头部AChE亚细胞分布结果表明，AChE活性主要分布在线粒体层，其他依次为微粒体层、胞质溶液以及细胞核和细胞碎片层。另外，对克隆得到的ace2片段（GenBank登录号: JQ359006）不同组织的相对表达量分析结果表明，头部ace2基因的相对表达量最高，胸部是头部的52%，腹部是头部的14%。以上研究结果表明，小峰熊蜂AChE主要分布于神经系统发达的头部，在亚细胞上主要存在于有膜的细胞器上。
对不同发育期小峰熊蜂AChE活性的测定及ace2基因的时间表达特性分析表明，小峰熊蜂幼虫期和蛹期AChE活性较低，出房后的工蜂头部AChE活性开始升高，5日龄，20日龄和30日龄AChE活性相对较高。进一步对ace2mRNA表达分析表明，幼虫期和蛹期ace2的表达量相对较低，表达量高峰出现在20日龄，其次是10日龄、5日龄和25日龄。据此可知，小峰熊蜂工蜂幼虫和蛹AChE活性及表达量均显著低于成年工蜂，而成年蜂之间无显著性差异，表明采集蜂和哺育蜂表达无显著性差异（P≥0.05）。
3、AChE离体抑制作用表明，辛硫磷、三唑磷、残杀威和丙溴磷对小峰熊蜂头部AChE离体抑制中浓度（IC_(50)）依次增加，抑制作用依次减弱。另外，生物测定结果表明此4种药剂对小峰熊蜂致死中浓度（LC_(50)）依次增加，毒力依次减弱，经此4种药剂亚致死剂量处理后的小峰熊蜂头部AChE活性均下降。说明有机磷和氨基甲酸酯类杀虫剂对小峰熊蜂AChE具有明显的剂量-效应关系，杀虫剂LC_(50)和IC_(50)变化趋势一致。
Bumblebee is one of the most crucial pollinators for the endangered, alpine plants and greenhousecrops. Bombus hypocrita (Hymenoptera: Apidae) is one of the dominant bumblebees in China, and iswidely used as one of the most crucial pollinators in greenhouse due to easy mass-rearing, strongpopulation and effective pollinating performance. However, the application of insecticides wasconsidered as one of the most reseaons for declining of the bumblebees. Organophosphate andcarbamate insecticides are the most widely used pesticides, which taget in acetylcholinesterase (AChE).In order to properly protect and utilize bumblebee, we studied AChE in Bombus hypocrita. We usedenzyme activity determination and real time PCR to indentify the spatial and temporal expressioncharacteristics of AChE in B. hypocrita. In addition, the inhibition and toxicities of severalorganophosphate and carbamate insecticides on B. hypocrita were conducted by in vitro inhibiton assaysand bioassays.
The results are as follows:
(1) Optimization of conditions for assaying activity of AChE in B. hypocrita. The results showed thatenzyme concentration0.25g protein/L, substrate concentration0.8mmol/L, pH7.5, reactiontemperature40℃, and reaction time5min were the optimal conditions. The five factors allextremely significantly impact the assaying of AChE in the head of B. hypocrita. The effect sizeswith the order are enzyme concentration, pH, temperature, substrate concentration and reactiontime.
(2) The distribution of AChE activity and ace2gene mRNA relative expression were conducted for thespatial expression characteristics. The tissue distribution results showed that the head AChEactivity (63.55%) was significantly higher than the thorax (18.44%) and abdomen (18.01%).Further study presented that the AChE in the head subcellular distribution of AChE activity wasmainly located in the mitochondrial layer, followed by the microsomal layer, and the solution ofcytoplasm and nucleus and cell debris layer. In addition, the results of the ace2(GenBankaccession: JQ359006) relative expression for the different tissues showed that the head ace2relative expression level was the highest. The thorax ace2expression was52%of the head,abdomen14%of the head. These results suggest that AChE is mainly distributed in the nervoussystem developed in the head of B. hypocrita, expecially in the subcellular membrane organelles.
The specific activity of AChE and ace2gene relative expression of different developmentalstages showed the temporal characteristics. The results showed during the developmental stages,AChE activity for larvae was the lowest and began to increase after emergence. During thedevelopmental stages, AChE activity for5thday,20thday and30thday were higher than others. Onthe other hand, AChE mRNA was detected lowly in the larvae and pupae stage of the workers byreal-time PCR method. The highest expression level was in the20thday old adult workers,following by the10th,5th, and25th. We make conclusion that the adult stage had the highest AChE specific activity and the highest ace2mRNA expression during the development of B. hypocrita,but no significant difference was found among the different ages of adults from0thday ofemergence to the30thday, which means that no significance were found between the foragers andnurses of B. hypocrita.
(3) In vitro inhibition assays showed that the inhibition concentration (IC_(50)) of phoxim, triazophos,propoxur and profenofos in turn weakened and the inhibiton were enhanced. In addition, bioassaysshowed that the lethal concentration (LC_(50)) of these four insecticides was enhanced and thetoxicities were weakened. After exposure to the sublethal dose of the four insecticides, the AChEactivities of the heads in B. hypocrita decreased compared to the control groups. We makeconclusion that organophosphate and carbamate insecticides on B. hypocrita have a significantdose-response relationship, and the trends of LC_(50)and IC_(50)are similar.
1、利用正交试验方法优化得到了小峰熊蜂AChE最佳测定条件。结果表明：酶浓度0.25g蛋白质/L，底物浓度0.8mmol/L，pH值7.5，温度40℃，反应时间5min为小峰熊蜂AChE的最佳测定条件。这5个因素对小峰熊蜂头部AChE活性的测定均有极显著的影响（P≤0.01），影响大小依次为酶浓度、pH、温度、底物浓度和反应时间。
2、对小峰熊蜂AChE活性分布的研究及ace2基因相对表达量的分析明确了小峰熊蜂AChE的空间表达特性。组织分布结果表明，小峰熊蜂头部AChE活性（63.55%）显著高于胸部（18.44%）和腹部（18.01%）（P≤0.05）。进一步对头部AChE亚细胞分布结果表明，AChE活性主要分布在线粒体层，其他依次为微粒体层、胞质溶液以及细胞核和细胞碎片层。另外，对克隆得到的ace2片段（GenBank登录号: JQ359006）不同组织的相对表达量分析结果表明，头部ace2基因的相对表达量最高，胸部是头部的52%，腹部是头部的14%。以上研究结果表明，小峰熊蜂AChE主要分布于神经系统发达的头部，在亚细胞上主要存在于有膜的细胞器上。
对不同发育期小峰熊蜂AChE活性的测定及ace2基因的时间表达特性分析表明，小峰熊蜂幼虫期和蛹期AChE活性较低，出房后的工蜂头部AChE活性开始升高，5日龄，20日龄和30日龄AChE活性相对较高。进一步对ace2mRNA表达分析表明，幼虫期和蛹期ace2的表达量相对较低，表达量高峰出现在20日龄，其次是10日龄、5日龄和25日龄。据此可知，小峰熊蜂工蜂幼虫和蛹AChE活性及表达量均显著低于成年工蜂，而成年蜂之间无显著性差异，表明采集蜂和哺育蜂表达无显著性差异（P≥0.05）。
3、AChE离体抑制作用表明，辛硫磷、三唑磷、残杀威和丙溴磷对小峰熊蜂头部AChE离体抑制中浓度（IC_(50)）依次增加，抑制作用依次减弱。另外，生物测定结果表明此4种药剂对小峰熊蜂致死中浓度（LC_(50)）依次增加，毒力依次减弱，经此4种药剂亚致死剂量处理后的小峰熊蜂头部AChE活性均下降。说明有机磷和氨基甲酸酯类杀虫剂对小峰熊蜂AChE具有明显的剂量-效应关系，杀虫剂LC_(50)和IC_(50)变化趋势一致。
Bumblebee is one of the most crucial pollinators for the endangered, alpine plants and greenhousecrops. Bombus hypocrita (Hymenoptera: Apidae) is one of the dominant bumblebees in China, and iswidely used as one of the most crucial pollinators in greenhouse due to easy mass-rearing, strongpopulation and effective pollinating performance. However, the application of insecticides wasconsidered as one of the most reseaons for declining of the bumblebees. Organophosphate andcarbamate insecticides are the most widely used pesticides, which taget in acetylcholinesterase (AChE).In order to properly protect and utilize bumblebee, we studied AChE in Bombus hypocrita. We usedenzyme activity determination and real time PCR to indentify the spatial and temporal expressioncharacteristics of AChE in B. hypocrita. In addition, the inhibition and toxicities of severalorganophosphate and carbamate insecticides on B. hypocrita were conducted by in vitro inhibiton assaysand bioassays.
The results are as follows:
(1) Optimization of conditions for assaying activity of AChE in B. hypocrita. The results showed thatenzyme concentration0.25g protein/L, substrate concentration0.8mmol/L, pH7.5, reactiontemperature40℃, and reaction time5min were the optimal conditions. The five factors allextremely significantly impact the assaying of AChE in the head of B. hypocrita. The effect sizeswith the order are enzyme concentration, pH, temperature, substrate concentration and reactiontime.
(2) The distribution of AChE activity and ace2gene mRNA relative expression were conducted for thespatial expression characteristics. The tissue distribution results showed that the head AChEactivity (63.55%) was significantly higher than the thorax (18.44%) and abdomen (18.01%).Further study presented that the AChE in the head subcellular distribution of AChE activity wasmainly located in the mitochondrial layer, followed by the microsomal layer, and the solution ofcytoplasm and nucleus and cell debris layer. In addition, the results of the ace2(GenBankaccession: JQ359006) relative expression for the different tissues showed that the head ace2relative expression level was the highest. The thorax ace2expression was52%of the head,abdomen14%of the head. These results suggest that AChE is mainly distributed in the nervoussystem developed in the head of B. hypocrita, expecially in the subcellular membrane organelles.
The specific activity of AChE and ace2gene relative expression of different developmentalstages showed the temporal characteristics. The results showed during the developmental stages,AChE activity for larvae was the lowest and began to increase after emergence. During thedevelopmental stages, AChE activity for5thday,20thday and30thday were higher than others. Onthe other hand, AChE mRNA was detected lowly in the larvae and pupae stage of the workers byreal-time PCR method. The highest expression level was in the20thday old adult workers,following by the10th,5th, and25th. We make conclusion that the adult stage had the highest AChE specific activity and the highest ace2mRNA expression during the development of B. hypocrita,but no significant difference was found among the different ages of adults from0thday ofemergence to the30thday, which means that no significance were found between the foragers andnurses of B. hypocrita.
(3) In vitro inhibition assays showed that the inhibition concentration (IC_(50)) of phoxim, triazophos,propoxur and profenofos in turn weakened and the inhibiton were enhanced. In addition, bioassaysshowed that the lethal concentration (LC_(50)) of these four insecticides was enhanced and thetoxicities were weakened. After exposure to the sublethal dose of the four insecticides, the AChEactivities of the heads in B. hypocrita decreased compared to the control groups. We makeconclusion that organophosphate and carbamate insecticides on B. hypocrita have a significantdose-response relationship, and the trends of LC_(50)and IC_(50)are similar.
引文
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