应用单克隆抗体评价捕食性天敌对麦长管蚜的控制作用
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摘要
采用目测法和网捕法对小麦整个生长季节内昆虫群落进行了系统调查,共查得麦田昆虫10目、62科、113种,其中植食类8目、18科、51种,捕食类5目、11科、23种,寄生类2目、12科、24种,腐食和吸血类1目、12科、15种。采用最优分割法分析了麦田昆虫群落的时间格局并提出了相应的防治策略。从营养层和功能团角度对麦田结构和动态进行了研究。
应用杂交瘤技术,在国内外首次以捕食麦长管蚜的多异瓢虫的消化道粗提物为免疫原,成功地制备出2株高度特异性的麦长管蚜单克隆抗体,分别命名为:EGA-4A9和EGA-2C4。应用间接ELISA测定抗体效价的结果表明,2株单克隆抗体的效价非常高都达到4.09×106以上。这2株抗体都不与麦田中其它种类的昆虫及捕食者发生交叉反应,而且也不与捕食其它蚜虫的多异瓢虫反应,具有高度的特异性。利用鼠源单克隆抗体亚类鉴定试剂盒(ClonotypingTM System/HRP)以双抗夹心ELISA方法进行检测,结果表明EGA-4A9和EGA-2C4分泌的单克隆抗体均为IgG2a亚类。SDS-聚丙烯酰胺凝胶电泳和Western blot印迹分析表明,单克隆抗体EGA-4A9与取食麦长管蚜后的多异瓢虫粗提物中21kDa和23kDa的多肽结合,单克隆抗体EGA-2C4与取食麦长管蚜后的多异瓢虫粗提物中20kDa、22kDa和26kDa的多肽结合。从结果可知,单克隆抗体的结合蛋白是麦长管蚜在瓢虫消化道内降解的中间产物。
对3种主要的酶联免疫吸附试验(ELISA)的比较研究表明,间接ELISA的灵敏度高于直接ELISA,分别为0.083μg/ml抗原蛋白和0.33μg/ml抗原蛋白,而双抗夹心ELISA的灵敏度最差,根本无法使用。在不同的温度和捕食量下,猎物在捕食者体内成单峰型的降解曲线。温度对猎物的可测定时间有显著影响,随着温度上升猎物的可测定时间不断缩短,特别是当温度达到30℃时,猎物的降解速率迅速上升,其可测定时间非常短,只有1.17天;而当温度在15℃～20℃之间时,猎物降解缓慢,可测定时间为3.52和3.10天。捕食量对猎物的可测定时间也有显著的影响,随着取食量的增加,猎物的可测定时间也随之延长,在25℃时捕食量为1头到3头时,可测定时间从2.80天延长到4.25天。
应用麦长管蚜单克隆抗体检测了内蒙古农业大学农场小麦田的捕食性天敌。结果表明,麦田的瓢虫种类较多,对麦长管蚜有较好的控制作用。其中多异瓢虫和龟纹瓢虫的种群数量多,阳性反应率较高,控制作用最好。捕食性天敌总的阳性反应随麦长管蚜种群密度的增加而增加相关关系显著(p<0.03)。
本文应用制备的单克隆抗体结合田间调查评价了麦田的主要捕食性天敌及其捕食作用,为有效的利用天敌控制害虫提供了一定的理论依据。
Systematic investigation and analysis of insect communities in wheat fields were carried out. The results indicated that there were 10 orders, 62 families, 113 species of insects, including 8 orders, 18 families, 51 species of phytophagous insects. 5 orders, 11 families, 23 species of predators; 2 orders, 12 families, 24 species of parasitoids; and 1 order, 12 families, 15 species of detritivores and bloodsuckers. Using the best sequent division method, the seasonal dynamics of insect communities was analysed in two years and the control strategies in different periods were given. Moreover, the seasonal dynamics and structure of insect community in wheat fields were reseached from nutrient class and guild view.
Two highly specific monoclonal antibodies, namly EGA-4A9 and EGA-2C4, were developed against M.avenae using hybridoma technique.The two McAbs were produced with the gut homogenate of Adonia variegate which had eaten M.avenae as immunogen and had high absorption values even they were diluted over 4.096×106 times.They did not cross-react with any other insects and predators tested. Moreover, they did not react with the A.variegate which had predated other aphids.These McAbs were tested with the methods of double antibody sandwich ELISA using SBA clonotrying system/HRP.The result showed that these two McAbs belonged to IgG2a subclass. SDS-PAGE and Western blot analysis indicated that McAb EGA-4A9 reacted with the polypeptides with molecular weight of 21 KDa and 23 KDa and MCAb EGA-2C4 reacted with the polypeptides with molecular weight of 20 KDa、22 KDa and 23KDa.The result indicated that these two McAb’s combining proteins were the middle products which M.avenae had digested in the gut of ladybird beetle,and they did not have the corresponding protein zone to the M.avenae antigen.
The comparasions of three types of ELISAs showed that an indirect ELISA was higher than a direct ELISA in sensitity and a double antibody sandwich ELISA was the lowest in sensitity. The sensitities of an indirect ELISA and a direct ELISA was 0.083 and 0.33μg/ml protein antigens, respectively, while a double antibody sandwich ELISA was too lowly sentitive to be able to be used. At the different temperature and meal size, the decayed curve of prey in the gut of predator became a single-peak mode which rose firstly and declined subsequently because the prey’s combining protein was the middle product with M.avenae had digested. Temperature had a great effect on prey detection periods. With the temperature increasing, prey detection periods decreased. Especially when the temperature was up to 30℃,the decay rate of prey increased sharply and the dectection period was very short, only 1.17 day. At the temperature rang of 15℃to 20℃,the decay rate of prey was slower than at 25℃and the detection periods prolonged from 3.10 to 3.52 days. The meal size also affected the detection period remarkably. While the number of prey which consumed by predators increased from one adult to three adults, the prey detection periods lengthened from 2.80 to 4.25 days at 25℃.
The monoclonal antibody EGA-4A9 was used to test the arthropod predators from the Inner Mongolla Agricultural University’s wheat field. The results indicated that there were great many kinds of ladybirds and they had a great control effect on the M.avenae.Among these natural enemies, the quantities of A.variegate and Propylea japonica were the largest and their positive rates were higher than others, so they had the better control effect on M.avenae.The total positive rate of predatory natural enemies inceased with the increase of the M.avenae density. Correlation analysis indicated that the total positive rate of predatory natural enemies had a significant relation to the density of M.avenae(p<0.03).
We evaluated the control effect of the major predatory natural enemies in the field using McAbs of M.avenae and investgation,and all these results provided the basis for the effective use of predatory natural enemies.
应用杂交瘤技术,在国内外首次以捕食麦长管蚜的多异瓢虫的消化道粗提物为免疫原,成功地制备出2株高度特异性的麦长管蚜单克隆抗体,分别命名为:EGA-4A9和EGA-2C4。应用间接ELISA测定抗体效价的结果表明,2株单克隆抗体的效价非常高都达到4.09×106以上。这2株抗体都不与麦田中其它种类的昆虫及捕食者发生交叉反应,而且也不与捕食其它蚜虫的多异瓢虫反应,具有高度的特异性。利用鼠源单克隆抗体亚类鉴定试剂盒(ClonotypingTM System/HRP)以双抗夹心ELISA方法进行检测,结果表明EGA-4A9和EGA-2C4分泌的单克隆抗体均为IgG2a亚类。SDS-聚丙烯酰胺凝胶电泳和Western blot印迹分析表明,单克隆抗体EGA-4A9与取食麦长管蚜后的多异瓢虫粗提物中21kDa和23kDa的多肽结合,单克隆抗体EGA-2C4与取食麦长管蚜后的多异瓢虫粗提物中20kDa、22kDa和26kDa的多肽结合。从结果可知,单克隆抗体的结合蛋白是麦长管蚜在瓢虫消化道内降解的中间产物。
对3种主要的酶联免疫吸附试验(ELISA)的比较研究表明,间接ELISA的灵敏度高于直接ELISA,分别为0.083μg/ml抗原蛋白和0.33μg/ml抗原蛋白,而双抗夹心ELISA的灵敏度最差,根本无法使用。在不同的温度和捕食量下,猎物在捕食者体内成单峰型的降解曲线。温度对猎物的可测定时间有显著影响,随着温度上升猎物的可测定时间不断缩短,特别是当温度达到30℃时,猎物的降解速率迅速上升,其可测定时间非常短,只有1.17天;而当温度在15℃～20℃之间时,猎物降解缓慢,可测定时间为3.52和3.10天。捕食量对猎物的可测定时间也有显著的影响,随着取食量的增加,猎物的可测定时间也随之延长,在25℃时捕食量为1头到3头时,可测定时间从2.80天延长到4.25天。
应用麦长管蚜单克隆抗体检测了内蒙古农业大学农场小麦田的捕食性天敌。结果表明,麦田的瓢虫种类较多,对麦长管蚜有较好的控制作用。其中多异瓢虫和龟纹瓢虫的种群数量多,阳性反应率较高,控制作用最好。捕食性天敌总的阳性反应随麦长管蚜种群密度的增加而增加相关关系显著(p<0.03)。
本文应用制备的单克隆抗体结合田间调查评价了麦田的主要捕食性天敌及其捕食作用,为有效的利用天敌控制害虫提供了一定的理论依据。
Systematic investigation and analysis of insect communities in wheat fields were carried out. The results indicated that there were 10 orders, 62 families, 113 species of insects, including 8 orders, 18 families, 51 species of phytophagous insects. 5 orders, 11 families, 23 species of predators; 2 orders, 12 families, 24 species of parasitoids; and 1 order, 12 families, 15 species of detritivores and bloodsuckers. Using the best sequent division method, the seasonal dynamics of insect communities was analysed in two years and the control strategies in different periods were given. Moreover, the seasonal dynamics and structure of insect community in wheat fields were reseached from nutrient class and guild view.
Two highly specific monoclonal antibodies, namly EGA-4A9 and EGA-2C4, were developed against M.avenae using hybridoma technique.The two McAbs were produced with the gut homogenate of Adonia variegate which had eaten M.avenae as immunogen and had high absorption values even they were diluted over 4.096×106 times.They did not cross-react with any other insects and predators tested. Moreover, they did not react with the A.variegate which had predated other aphids.These McAbs were tested with the methods of double antibody sandwich ELISA using SBA clonotrying system/HRP.The result showed that these two McAbs belonged to IgG2a subclass. SDS-PAGE and Western blot analysis indicated that McAb EGA-4A9 reacted with the polypeptides with molecular weight of 21 KDa and 23 KDa and MCAb EGA-2C4 reacted with the polypeptides with molecular weight of 20 KDa、22 KDa and 23KDa.The result indicated that these two McAb’s combining proteins were the middle products which M.avenae had digested in the gut of ladybird beetle,and they did not have the corresponding protein zone to the M.avenae antigen.
The comparasions of three types of ELISAs showed that an indirect ELISA was higher than a direct ELISA in sensitity and a double antibody sandwich ELISA was the lowest in sensitity. The sensitities of an indirect ELISA and a direct ELISA was 0.083 and 0.33μg/ml protein antigens, respectively, while a double antibody sandwich ELISA was too lowly sentitive to be able to be used. At the different temperature and meal size, the decayed curve of prey in the gut of predator became a single-peak mode which rose firstly and declined subsequently because the prey’s combining protein was the middle product with M.avenae had digested. Temperature had a great effect on prey detection periods. With the temperature increasing, prey detection periods decreased. Especially when the temperature was up to 30℃,the decay rate of prey increased sharply and the dectection period was very short, only 1.17 day. At the temperature rang of 15℃to 20℃,the decay rate of prey was slower than at 25℃and the detection periods prolonged from 3.10 to 3.52 days. The meal size also affected the detection period remarkably. While the number of prey which consumed by predators increased from one adult to three adults, the prey detection periods lengthened from 2.80 to 4.25 days at 25℃.
The monoclonal antibody EGA-4A9 was used to test the arthropod predators from the Inner Mongolla Agricultural University’s wheat field. The results indicated that there were great many kinds of ladybirds and they had a great control effect on the M.avenae.Among these natural enemies, the quantities of A.variegate and Propylea japonica were the largest and their positive rates were higher than others, so they had the better control effect on M.avenae.The total positive rate of predatory natural enemies inceased with the increase of the M.avenae density. Correlation analysis indicated that the total positive rate of predatory natural enemies had a significant relation to the density of M.avenae(p<0.03).
We evaluated the control effect of the major predatory natural enemies in the field using McAbs of M.avenae and investgation,and all these results provided the basis for the effective use of predatory natural enemies.
引文
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