【作者】 刘召；

【导师】 花保祯； Walter S. Leal；

【作者基本信息】 西北农林科技大学 ， 农业昆虫与害虫防治， 2011， 博士

【摘要】 脐橙螟蛾Amyelois transitella (Walker)是美国加利福尼亚州开心果、杏仁、核桃的重要经济害虫,以幼虫蛀食坚果果肉,在生产和储藏过程中均可危害,常造成严重的经济损失。由于其隐蔽的取食特性,该害虫难以防治,目前的防治手段仅限于果园内农事操作及喷洒化学药剂进行虫口压制。研究A. transitella的化学感受机制,阐明其与寄主植物之间的相互关系,探索新的化学生态学防治手段,对于A. transitella的控制有着重要的理论和实践意义。本文首先表达并纯化了A. transitella两种普通气味结合蛋白AtraGOBP1和AtraGOBP2,研究了这两种蛋白与植物源挥发物的作用机制。同时,利用电生理手段(触角电位仪技术)测定了A. transitella化学感受的电生理反应,并对A. transitella化学感受机制进行了探讨。在此基础上,在室内和田间检测了由这些挥发气味物质制成的诱捕器对该害虫产卵行为的影响,为利用这些化学挥发气味物质进行害虫的检测和防治进行了基础性研究。主要结果如下：1.利用LB培养基在转入AtraGOBP1和AtraGOBP2基因的大肠杆菌Escherichia coli中成功的表达了AtraGOBP1和AtraGOBP2蛋白。LB培养基中的大肠杆菌细胞经过高速离心后并在液氮中经三次冻融裂解细胞,利用Tris-HC1缓冲液提取目标蛋白,高速离心去除细胞残体,在蛋白快速纯化仪(FPLC)上用DEAE, Q-Seph, MONO Q, gel filtration等柱子成功纯化到这两种蛋白的高纯度(>98%)蛋白晶体。2.用高效液相色谱-电喷雾串联质谱(LC-ESI-MS)检测方法对AtraGOBP1和AtraGOBP2的结构研究表明：AtraGOBP1分子量为16897 Da(理论计算值为16903 Da), AtraGOBP2分子量为16161 Da(理论计算值为16166 Da)。这两种蛋白分别失去6个氢原子,使实际检测到的分子量比由氨基酸序列计算得到的理论值减小6 Da,说明这两种蛋白都进行了正确的折叠,分别合成了3对二硫键结构。利用圆二色谱仪(Circular Dichroism)检测发现AtraGOBP1和AtraGOBP2在193、209、223 nm处各出现一个波峰(谷),证明这两种蛋白与其它气味结合蛋白一样,都富含α螺旋结构；同时发现在pH7和pH 5.5的缓冲液中蛋白构象存在差异。3.利用cold binding assay法检测发现：在pH 7情况下,两种普通气味结合蛋白AtraGOBP1及AtraGOBP2均能与植物源挥发物decanal和nonanal结合；而在pH 5和缓冲液对照中均没有发生结合。但对于linalool、1-phenylethanol、PAPE、IBAPE等化合物,由于在pH 5和对照中存在较高的背景,难以得出明确结论。为确定这些化合物能否与AtraGOBP1和AtraGOBP2结合,我们采用了fluorescence binding assay检测,结果表明,AtraGOBP1及AtraGOBP2能与decanal、nonanal发生明显的结合,而与linalool、1-phenylethanol、PAPE、IBAPE则没有明显的结合。4.利用触角电位仪检测成虫触角发现：decanal、nonanal、linalool、1-phenylethanol、PAPE、IBAPE均能引发A. transitella雌、雄成虫的触角电生理反应,反应强度随着化合物浓度的升高而增大。对不同龄期的成虫触角对所测定的植物源挥发物的敏感度观察表明：在第0到4天中成虫触角对所测定的植物源挥发物均有较高的敏感度,而在第5天触角敏感度则明显降低。与气味结合蛋白与植物源挥发物结合试验比较表明,有其它气味结合蛋白参与信息素从外界环境到昆虫体内受体的转运。5.为检测各种植物源挥发物质对成虫产卵行为的影响,将不同剂量(10μl x 10μg/μl,50μl x 10μg/μl,100μl xμl的待测植物源挥发物质的己烷溶液滴在滤纸上制成诱饵,并用湿润卫生纸保湿；然后将诱饵及交配后的15头雄成虫及10头雌成虫放入(60×60×60cm)的尼龙网中观测成虫产卵行为,结果表明linalool和1-phenylethanol在3种剂量下均能引诱A. transitella产卵,其余植物源气味挥发物质对产卵没有明显的诱集效果。大田生测结果表明：在50μl x 10μg/μl,100μl x 10μg/μl两个剂量下,linalool和1-phenylethanol在能引诱A. transitella产卵,而10μl x 10μg/μl剂量下,则不能诱发脐橙螟蛾产卵；而其它的植物源挥发物在所测定的剂量条件下,也不能诱发该害虫产卵。更多还原

【Abstract】 The navel orangeworm, Amyelois transitella (Walker), is an important pest on pistachio, almonds, and walnuts in California, USA. It bores into and feeds the nuts both in the orchard and in the process of producing and store, frequently causing serious damage. It is difficult to control because of the bore-feeding characteristic. Now, the population control method is only by orchard cleaning after harvest and insecticide spraying when the insect population is high. Therefore, it is important to study the semiochemical detection mechanism, to identify the relationship between the insect and its hosts, to explore insect chemical ecological method for successful A. transitella population management.Here, two general odorant binding proteins from A. transitella, AtraGOBP1 and AtraGOBP2, were expressed and purified, and the interactions between these two GOBPs and plant volatile chemicals were also studied. At the same time, the electrical response of A. transitella antennae elicited by plant volatile chemicals was examined with electroantennography, and the perception mechanism was also discussed. Based on these researches, the effect of traps made with plant volatile chemicals on oviposition behavior was tested in both room and field to use these chemicals to monitor and control this pest. The main results are shown as follows:1. AtraGOBP1 and AtraGOBP2 protein were expressed and purified successfully in the transformed bacteria, Escherichia coli, which contains the project protein DNA (AtraGOBPl or AtraGOBP2 gene) and incubated in LB medium. The bacteria was harvested by centrifuging at 4500×g, followed by three times of freeze and thaw performance to cause lesion in the bacterial membrane. The protein was extracted with Tris-HCl buffer which followed by high speed centrifuge to remove the cell debris. High purity protein was got after the proteins solution passing through FPLC equipped with DEAE, Q-Seph, MONO Q, and gel filtration columns.2. The results from LC-ESI-MS analysis show that AtraGOBPl gave a molecular mass of 16,897 Da consistent with the calculated molecular mass of 16,903 Da given the formation of three disulfide bridges (expected 16,897 Da). Deconvolution of the mass spectrum from AtraGOBP2 gave a molecular mass of 16,161 Da in close agreement with the calculated molecular mass of 16,166 Da considering the formation of three disulfide linkages (expected, 16,160 Da). Far-ultraviolet circular dichroism spectra from both proteins resembled that of the pheromone-binding protein AtraPBP1, with maximum at ca.193 nm and two minima at 209 and 223 nm. Therefore, both AtraGOBP1 and AtraGOBP2 areα-helical-rich proteins, which is a common feature of moth OBPs. And they experienced a pH-dependence structure conformation change from pH 5.5 to pH 7.3. With cold binding assay method, it was found that both AtraGOBP1 and AtraGOBP2 were able to bind with decanal and nonanal at pH 7, while no obvious binding was detected at pH 5 or in control. For the rest plant volatile chemical, linalool,1-phenylethanol, PAPE and IBAPE, it is difficult to draw conclusion because the high background levels at pH 5 and the control. Competitive fluorescence binding with NPN as a reporter was performed to examine the binding ability of these chemical with these to GOBPs. The results showed that decanal and nonanal displaced NPN at pH 7, in contrast no displacement was found at pH 5 or in the control. None of the rest tested ligands displaced NPN, which means these two GOBPs were unable to bind with these chemicals.4. EAG recordings from live moths showed that both male and female antennae responded to these plant volatile chemicals in a dose-dependent manner. Experiments were also conducted to determine the effect of age on EAG response of female and female to plant volatile chemicals. The result showed that 0 to 4 days old male and female were sensitive to the tested plant volatile chemicals, and there were an obvious sensitivity decrease on day-5 in contrast with previous stages. Combined the information from interaction of the tested ligands and the GOBPs, we got the conclusion that there should be more odorant binding proteins were involved in the semiochemicals transport from environment to odor receptors.5. With the trap made with plant volatile chemical on filter papers with soaked paper towel to keep humidity, the effect of these chemicals on A. transitella oviposition behavior was examined at three different doses (10μl×10μg/μl,50μl×10μg/μl,100μl×10μg/μl). Then 15 males and 10 females were pun inside the nylon cage (60×60×60 cm) to examine the oviposition behavior. The result showed that both linalool and 1-phenylethanol attracted the female to lay egg on the trap. No eggs were found on the trap of other plant volatile chemicals. The bioassay in the field showed that:eggs of A. transitella were found on the trap made with linalool and 1-phenylethanol at dose of 50μl×10μg/μl,100μl×10μg/μl. However, no eggs were found on the trap with dose 10μl×10μg/μl. At the same condition, no eggs were found on the trap of other plant volatile chemicals at all the tested doses.更多还原