【作者】 代平礼；

【导师】 周婷； 孙继虎；

【作者基本信息】 中国农业科学院 ， 特种经济动物饲养， 2011， 博士

【摘要】 蜜蜂是公认的有益昆虫,具有经济和生态重要性,既能提供有益的蜂产品又能为植物授粉。对提高农作物产量、改善产品品质和维持生态平衡具有十分重要的作用。近年来世界范围内蜜蜂大量减少,杀虫剂中毒是导致蜂群下降的主要原因之一。转基因抗虫植物的发展,为害虫防治提供了一条新思路,减少了杀虫剂的使用。新型杀虫蛋白基因cry1Ah基因是中国农业科学院植物保护研究所从Bt菌株BT8中鉴定克隆的,其编码蛋白对鳞翅目害虫具有强毒力,尤其对亚洲玉米螟(Ostrinia furnacalis)的毒力强于目前使用的cry1A类基因。转cry1Ah基因基因抗虫玉米具有很好的应用前景。因此,开展转cry1Ah基因玉米对蜜蜂的安全性研究很有必要。本文从农业生态系统的角度出发通过毒蛋白的实验室测验、Bt玉米花粉饲喂蜂群幼虫试验和更接近实际农业生态条件的大田网室试验,测定转Bt cry1Ah基因玉米对蜜蜂的直接影响;并测定了Cry1Ah毒素对蜜蜂敌害大蜡螟的毒性以及螨害蜜蜂对毒素敏感性的改变,并且以一些化学杀虫剂为阳性对照研究其对蜜蜂的致死亚致死效应。具体结果如下:1、在实验室将接近Bt玉米花粉中表达量浓度1 ng/ml、10 ng/ml和不现实的高浓度10000 ng/ml Cry1Ah毒素混入糖水中饲喂蜜蜂工蜂,测定Cry1Ah毒素对意大利蜜蜂和中华蜜蜂存活、寿命、花粉取食量、王浆腺重量和学习行为的影响。结果显示三种浓度Cry1Ah毒素对意大利蜜蜂和中华蜜蜂工蜂的存活、寿命、花粉取食量和王浆腺重量未见不利影响。取食含10000 ng/ml Cry1Ah毒素糖水的意大利蜜蜂对30%蔗糖水的PER响应水平较对照低,而且对学习行为有不利影响。2、通过解剖工蜂大脑,培养获得大量分离的蜜蜂脑神经细胞,利用膜片钳技术全细胞记录模式研究Bt毒素对蜜蜂脑神经细胞兴奋性的影响。Bt毒素在10ng/ml和100ng/ml浓度下短时间(3-5min)条件下对蜜蜂脑神经元的阈电流没有显著改变,表明对脑神经细胞兴奋性没有显著影响。3、给意大利蜜蜂和中华蜜蜂蜂群中4-6日龄幼虫饲喂转基因玉米花粉、常规玉米花粉、杂花粉、以及哺育蜂饲喂,转基因玉米花粉对封盖率、出房率、发育成功率和发育历期没有显著影响,表明转cry1Ah基因玉米花粉对意大利蜜蜂和中华蜜蜂幼虫的存活和发育没有不良影响。以联苯菊酯和溴氰菊酯作为阳性对照,通过饲喂的方式测定了这两种杀虫剂对蜜蜂的亚致死效应,结果表明联苯菊酯和溴氰菊酯显著降低了发育成功率,延长了发育历期。4、通过大田网室试验测定在实际田间条件下转cry1Ah基因玉米对意大利蜜蜂的风险。意大利蜜蜂蜂群在扬花期分别放入种植转基因玉米和常规玉米的网室中,测定对蜂群存活、发育和行为的影响。结果显示,Bt玉米对发育历期、工蜂存活、蜜蜂体重、王浆腺重量、群势、采集行为和嗅觉学习行为均无不利影响。5、通过饲喂含毒素糖水的方法在实验室探讨了Cry1Ah毒素对螨害蜜蜂的影响。1 ng/ml、10 ng/ml和10000 ng/ml三种浓度Cry1Ah毒素对螨害蜜蜂的存活没有显著影响。与此同时开展了Cry1Ah毒素对蜜蜂敌害大蜡螟(Galleria mellonella)的致死效应研究。结果表明大蜡螟幼虫取食含有Cry1Ah毒素的人工饲料,其死亡率较对照组高。Cry1Ah毒素可作为一种可能的防治大蜡螟的措施。更多还原

【Abstract】 The honeybee is widely recognized as a beneficial insect of agronomic, ecological, and scientific importance. It produces commercially valuable products (honey, pollen, royal jelly, propolis, and wax) and plays a major role in crop pollination. Insecticide is one of causes of honeybee colony disorder in the world. Transgenic insect-resistant crop is one of the possible ways forward with the aim of combining higher yield and quality with environmentally friendly agronomic practices. Bt crop truly reduces insecticide applications, then Bt crops may increase the abundance of honeybees. The cry1Ah gene was one of the novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8 which exhibited high toxicity against lepidopteran larvae. Cry1Ah protein showed higher toxicity to Asian corn borer (Ostrinia furnacalis) than that of any other cry1A genes. The cry1Ah gene was a candidate gene for insect resistant transgenic corn research. Assessment of impacts on honeybee is an essential part of the risk assessment process for Bt cry1Ah gene corn.The objective of this study was to assess effects of various concentrations of the Cry1Ah toxin in laboratory condition and Bt corn pollen under field cage conditions on honeybees. The effects of Cry1Ah toxin on wax moth (Galleria mellonella) and honeybees with parasitical mites were also tested. The lethal and sublethal effects of chemical insecticides on honeybees were assessed as positive reference. The results were as follows:1. We conducted feeding trials in a laboratory setting to test for possible adverse effects of different concentrations (10000 ng/ml, 10 ng/ml and 1 ng/ml) of microbially produced Cry1Ah toxin on survival, pollen consumption and hypopharyngeal gland mass of Apis mellifera ligustica and Apis cerana cerana. There were no significant differences in mortalities of Apis mellifera ligustica or Apis cerana cerana among groups fed sugar syrup with or without Cry1Ah toxin within 24, 48 or 72h. Adverse effects of Cry1Ah toxin on survival of Apis mellifera ligustica and Apis cerana cerana were not observed. No significant differences were found in the longevity of Apis mellifera ligustica fed sugar syrup with Cry1Ah toxin compared with control. In experiments with Apis cerana cerana fed Cry1Ah toxin, only a concentration of 10ng/ml Cry1Ah toxin resulted in a significantly longer longevity compared with the control. No differences were detected for pollen consumption and hypopharyngeal gland mass of Apis mellifera ligustica and Apis cerana cerana.2. We dissociated neurons from brains of bee workers. This preparation resulted in a large number of isolated viable neurons. Effect of Bt toxin on the excitability of the isolated neurons were observed by the whole-cell configuration of the patch-clamp technique. The results showed that Bt toxin (10ng/ml and 100ng/ml) did not affect excitability of the isolated neurons, because current threshold did not change significantly.3. The effects of dietary transgenic Bt corn pollen on honeybee worker larvae of Apis mellifera ligustica and Apis cerana cerana were examined. We measured cap rate, emergence rate, success rate of development, and immature stage after 4-6-day-old larvae were fed various pollens (Bt cry1Ah gene corn pollen, non-Bt corn pollen, mixed bee pollen and control). There were no significant differences in all the parameters tested among treatments. Our studies suggest that transgenic Bt corn pollen does not pose a threat to honeybee larval development. The sublethal effects of chemical insecticides bifenthrin and deltamethrin on honeybees were assessed as positive reference. Effects of two pesticides at the sub-lethal concentration on honeybees were examined with the feeding method. It was shown that both bifenthrin and deltamethrin significantly decreased the rate for bee to develop to adulthood and increased their immature periods.4. Honeybee could be exposed to insecticidal proteins from transgenic plants via pollen. Assessment of impacts on honeybee is an essential part of the risk assessment process for Bt corn. A field trial was conducted to evaluate the effect of transgenic Bt cry1Ah gene corn on honeybee, Apis mellifera ligustica. Colonies of honeybees were moved to Bt or non-Bt corn fields during bloom and then sampled for survival, development and behavior. No differences in immature stages, workers survival, bee body weight, hypopharyngeal glands weight, colony performance, foraging activity and olfactory learning abilities were detected between colonies placed in non-Bt corn fields and those in Bt corn fields. Results from this study suggest that transgenic Bt cry1Ah gene corn pollen does not have adverse effects on Apis mellifera ligustica.5. The effects of dietary Bt Cry1Ah toxin (1 ng/ml、10 ng/ml and 10000 ng/ml) on honeybee workers parasited by Varroa destructor were examined. There were no significant differences in mortality tested between workers fed Bt Cry1Ah toxin and control. We also evaluated Bt Cry1Ah toxin as a potential control for wax moth larvae in a laboratory study. We fed wax moth larvae three concentration of Cry1Ah toxin: 1 ng/g、10 ng/g and 10000 ng/g. We found that the mortality of larvae fed artificial diet containing Cry1Ah toxin was significantly greater than the mortality of larvae fed artificial diet without Cry1Ah toxin. Our studies suggest that transgenic Bt corn pollen does not pose a threat to honey bee larval development and has the potential to serve as an alternative control for wax moth.更多还原