杀虫蛋白在Bt水稻—植食者—蜘蛛食物链中的传递规律及对稻田代表性蜘蛛的影响
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摘要
1杀虫蛋白在Bt水稻—植食者—天敌食物网中的传递规律
综合应用机动吸虫器和扫网法于Bt稻田采集节肢动物,鉴定后进行ELISA检测Cry1Ab蛋白含量。结果表明在大多数植食性昆虫(如黑尾叶蝉、褐飞虱等)体内检测到了Cry1Ab蛋白,而且在多种寄生蜂和捕食性天敌体内也能检测到。这说明Cry1Ab可以通过植食者传递至更高的营养层。天敌对Cry1Ab的取食有可能是直接取食Bt水稻,也有可能是取食了其他体内含有Cry1Ab蛋白的节肢动物。不同节肢动物暴露于Cry1Ab蛋白的程度,在将来对非靶标节肢动物进行安全评价时应该作为参考。
2转Bt基因水稻对拟环纹豹蛛生长发育及免疫的影响及其机理的研究
比较分析Bt水稻对拟环纹豹蛛生长发育和繁殖能力的影响。结果表明,Bt水稻对拟环纹豹蛛的存活率、若蛛历期和繁殖力都没有显著影响。通过对拟环纹豹蛛血细胞数量、包囊率和PO活性的检测,虽然杀虫剂显著降低了拟环纹豹蛛的血细胞数量和包囊率,但Bt水稻未对拟环纹豹蛛的免疫功能产生显著的影响。BBMV和Cry1Ab的结合实验表明拟环纹豹蛛的BBMV上没有Cry1Ab蛋白的受体,这可能是拟环纹豹蛛不受Bt水稻影响的原因所在。
3转Bt基因水稻对拟环纹豹蛛控害能力和功能反应的影响
采用ELISA和PCR方法检测了拟环纹豹蛛中肠褐飞虱的阳性率。结果表明,拟环纹豹蛛的褐飞虱平均阳性率为40%,Bt稻田和对照稻田之间没有显著差异。功能反应的研究结果表明,来自Bt稻田和对照稻田的拟环纹豹蛛在同一猎物密度下,对褐飞虱的捕食量无显著差异,且它们的功能反应同属于HollingⅡ型反应，瞬时攻击率和处理时间都无显著差异。
4用于评价蜘蛛猎物结构的LDR芯片制备及其初步应用
我们首次介绍了一种新方法,基于LDR可对同时追踪蜘蛛中肠15种猎物的灵敏有效的方法。我们验证了它的高特异性和高灵敏度。我们还进行了田间试验,对不同的蜘蛛中肠内容物进行分析可以追踪它们猎物的多样性。来自Bt稻田和对照稻田的数百个蜘蛛样品支持了该技术的可靠性。这些结果意味着不仅在转基因水稻领域,在其他领域如外来生物入侵也可灵活的应用PCR-LDR进行捕食者和猎物之间的生态学研究。
1 Biotransfer and bioaccumulation of CrylAb protein in rice plant-herbivores-natural enemy food web
Arthropods were collected from Bt rice field by vacuum-suction machine and the sweep net method. After identification of arthropods species, ELISA was conducted to detecte the CrylAb level. The results showed that the majority of herbivorous insects (such as leafhopper, brown planthopper, etc.) contain significant quantities of CrylAb protein. Furthermore, parasitoid and predator collected from these agroecosystems also contained significant quantities of CrylAb protein indicating its movement into higher trophic levels. This uptake by natural enemy is likely to have occurred by direct feeding on plant material or the consumption of arthropod prey which contained these proteins. These levels of exposure should therefore be considered during future risk assessments of transgenic crops to nontarget herbivores and arthropod predators.
2 Effect of Bt rice on the development and immunity of Pardosa pseudoannulata and its mechanism
Assessment of the effect of Bt rice on growth and fecundity of P. pseudoannulata was conducted. The results show that, Bt rice did not significantly affect the survival rate, duration and fecundity of P. pseudoannulata. Furthermore the number of hemocytes, encapsulation rate and PO activity of P. pseudoannulata was detected. Although pesticides significantly reduced the number of hemocytes and encapsulation rate of P. pseudoannulata, Bt rice did not significantly impact immune function. BBMV and Cry1Ab binding experiments indicate the BBMV of P. pseudoannulata has no CrylAb protein receptor, which may be the reason why no negative effects of Bt rice on this spider.
3 Effect of Bt rice on the control pests ability and functional response of P. pseudoannulata
Percentage of P. pseudoannulata testing positive for Nilaparvata lugens remains under field condition was tested using ELISA and PCR. The results indicate the average percentage of P. pseudoannulata testing positive for N. lugens was 40%. No significant difference was found between Bt rice and control rice field. Functional response results show that P. pseudoannulata from Bt rice and control rice fields prey no significantly different amount N. lugens at the same prey density. Their functional response all belong to HollingⅡtype reaction, and the attack constant and handling time were not significantly different.
4 Preparation and preliminary application of LDR chip used to evaluate the structure of spider prey
For the first time, we describe a novel method, an available and sensitively approach to track 15 preys in the gut of spider, based on ligase reaction detection (LDR). We present estimators for several detectable parameters (sensitivity and specificity). We also describe field experiments that different spider species gut content analysis can track their predation diversity. Hundreds samples from the Bt rice and non-Bt control support the reliability of this technology. Finally, a total of 50 spiders for one sampling, in which way the predation diversity of spider could be observed, are suggested by authors. These results imply that PCR-LDR can be feasible and flexible applied to large scale ecological studies between predators and preys not only in transgenic rice field, but also in many other areas such as exotic biological invasion.
综合应用机动吸虫器和扫网法于Bt稻田采集节肢动物,鉴定后进行ELISA检测Cry1Ab蛋白含量。结果表明在大多数植食性昆虫(如黑尾叶蝉、褐飞虱等)体内检测到了Cry1Ab蛋白,而且在多种寄生蜂和捕食性天敌体内也能检测到。这说明Cry1Ab可以通过植食者传递至更高的营养层。天敌对Cry1Ab的取食有可能是直接取食Bt水稻,也有可能是取食了其他体内含有Cry1Ab蛋白的节肢动物。不同节肢动物暴露于Cry1Ab蛋白的程度,在将来对非靶标节肢动物进行安全评价时应该作为参考。
2转Bt基因水稻对拟环纹豹蛛生长发育及免疫的影响及其机理的研究
比较分析Bt水稻对拟环纹豹蛛生长发育和繁殖能力的影响。结果表明,Bt水稻对拟环纹豹蛛的存活率、若蛛历期和繁殖力都没有显著影响。通过对拟环纹豹蛛血细胞数量、包囊率和PO活性的检测,虽然杀虫剂显著降低了拟环纹豹蛛的血细胞数量和包囊率,但Bt水稻未对拟环纹豹蛛的免疫功能产生显著的影响。BBMV和Cry1Ab的结合实验表明拟环纹豹蛛的BBMV上没有Cry1Ab蛋白的受体,这可能是拟环纹豹蛛不受Bt水稻影响的原因所在。
3转Bt基因水稻对拟环纹豹蛛控害能力和功能反应的影响
采用ELISA和PCR方法检测了拟环纹豹蛛中肠褐飞虱的阳性率。结果表明,拟环纹豹蛛的褐飞虱平均阳性率为40%,Bt稻田和对照稻田之间没有显著差异。功能反应的研究结果表明,来自Bt稻田和对照稻田的拟环纹豹蛛在同一猎物密度下,对褐飞虱的捕食量无显著差异,且它们的功能反应同属于HollingⅡ型反应，瞬时攻击率和处理时间都无显著差异。
4用于评价蜘蛛猎物结构的LDR芯片制备及其初步应用
我们首次介绍了一种新方法,基于LDR可对同时追踪蜘蛛中肠15种猎物的灵敏有效的方法。我们验证了它的高特异性和高灵敏度。我们还进行了田间试验,对不同的蜘蛛中肠内容物进行分析可以追踪它们猎物的多样性。来自Bt稻田和对照稻田的数百个蜘蛛样品支持了该技术的可靠性。这些结果意味着不仅在转基因水稻领域,在其他领域如外来生物入侵也可灵活的应用PCR-LDR进行捕食者和猎物之间的生态学研究。
1 Biotransfer and bioaccumulation of CrylAb protein in rice plant-herbivores-natural enemy food web
Arthropods were collected from Bt rice field by vacuum-suction machine and the sweep net method. After identification of arthropods species, ELISA was conducted to detecte the CrylAb level. The results showed that the majority of herbivorous insects (such as leafhopper, brown planthopper, etc.) contain significant quantities of CrylAb protein. Furthermore, parasitoid and predator collected from these agroecosystems also contained significant quantities of CrylAb protein indicating its movement into higher trophic levels. This uptake by natural enemy is likely to have occurred by direct feeding on plant material or the consumption of arthropod prey which contained these proteins. These levels of exposure should therefore be considered during future risk assessments of transgenic crops to nontarget herbivores and arthropod predators.
2 Effect of Bt rice on the development and immunity of Pardosa pseudoannulata and its mechanism
Assessment of the effect of Bt rice on growth and fecundity of P. pseudoannulata was conducted. The results show that, Bt rice did not significantly affect the survival rate, duration and fecundity of P. pseudoannulata. Furthermore the number of hemocytes, encapsulation rate and PO activity of P. pseudoannulata was detected. Although pesticides significantly reduced the number of hemocytes and encapsulation rate of P. pseudoannulata, Bt rice did not significantly impact immune function. BBMV and Cry1Ab binding experiments indicate the BBMV of P. pseudoannulata has no CrylAb protein receptor, which may be the reason why no negative effects of Bt rice on this spider.
3 Effect of Bt rice on the control pests ability and functional response of P. pseudoannulata
Percentage of P. pseudoannulata testing positive for Nilaparvata lugens remains under field condition was tested using ELISA and PCR. The results indicate the average percentage of P. pseudoannulata testing positive for N. lugens was 40%. No significant difference was found between Bt rice and control rice field. Functional response results show that P. pseudoannulata from Bt rice and control rice fields prey no significantly different amount N. lugens at the same prey density. Their functional response all belong to HollingⅡtype reaction, and the attack constant and handling time were not significantly different.
4 Preparation and preliminary application of LDR chip used to evaluate the structure of spider prey
For the first time, we describe a novel method, an available and sensitively approach to track 15 preys in the gut of spider, based on ligase reaction detection (LDR). We present estimators for several detectable parameters (sensitivity and specificity). We also describe field experiments that different spider species gut content analysis can track their predation diversity. Hundreds samples from the Bt rice and non-Bt control support the reliability of this technology. Finally, a total of 50 spiders for one sampling, in which way the predation diversity of spider could be observed, are suggested by authors. These results imply that PCR-LDR can be feasible and flexible applied to large scale ecological studies between predators and preys not only in transgenic rice field, but also in many other areas such as exotic biological invasion.
引文
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