昆虫NPV垂直传递和PCR检测的研究
摘要
昆虫核型多角体病毒(NPV)是一种重要的病原微生物和生物杀虫剂，不仅具有专一性强、对环境和人畜安全、不杀伤天敌等优点，而且在其应用后能在自然界中长期存在，从而引起病毒流行病的大发生，可在较长时间内自然控制害虫种群密度的增长，在害虫的持续控制研究中发挥着重要的作用。
本文对室内保存的病毒株系HaNPV的毒力进行生物测定并根据实验结果计算出LC30、LC50、LC70和LC90。用上述4种浓度感染健康的棉铃虫3龄幼虫，分别收集感染未致死的棉铃虫蛹进行传代饲养,对不同处理的各代间幼虫的死亡率、化蛹率、羽化率和平均单头蛹重等生物学特性参数进行了研究和分析，并用PCR方法对传代棉铃虫的蛹和卵进行检测，从而对NPV室内垂直传递作了比较系统和详细的研究。实验结果如下：
(1)实验室保存病毒株系HaNPV的致死中浓度（LC50）和致死中量（LD50）分别为3.44×104PIB/ml和1995PIB。当用1.0×107 PIB/ml的浓度感染棉铃虫3龄幼虫时，其感染半致死时间（LT50）为4.77天，当浓度为1.0×106PIB/ml时，LT50为5.73天，用1.0×105PIB/ml的浓度感染时为6.63天。
（2）用LC70、LC50和LC30的浓度感染寄主时，各处理的子一代（F1）和子二代（F2）的幼虫死亡率分别为15～50.42%、21.67～47.92%和12.22～34.44%，均远高于对照的7.50～8.33%；在子三代（F3）和子四代（F4）中，除了处理LC30在子三代（F3）中的幼虫死亡率（17.78%）明显高于对照（8.83%）外，其它处理与对照均无明显差异。
（3）在幼虫化蛹率方面，亲代（F0）和子一代（F1）与对照之间存在明显差异。处理LC70、LC50和LC30的幼虫化蛹率分别为61.37～72.28%、77.31～87.36%和77.39～87.05%，处理LC90亲代（F0）的化蛹率仅为32.14%，均低于对照的93.69～96.63%；在子二代（F2）、子三代（F3）和子四代（F4）中，各处理与对照均差异不明显。
（4）处理LC90、LC70、LC50和LC30的亲代和子代在羽化率方面也存在差异。
在F0代中，处理LC90和LC70的羽化率分别为50.00%和51.52%，明显低于对照的85.99%；F1代中，处理LC70的羽化率（67%）也远低于对照（83.93%），其它处理与对照差异不明显；而在F2、F3和F4代中，处理和对照差异不明显。
（5）各处理在平均单头蛹重方面与对照也有明显的差异，在亲代（F0）中，各处理（0.1996～0.2356g）与对照（0.3036g）之间差异极显著 。在子一代（F1）和子二代（F2）中，处理LC70和LC50与对照之间差异极显著，其它处理与对照无差异。各处理在子三代（F3）和子四代（F4）代中与对照无差异。
（6）在参考其它昆虫NPV多角体蛋白基因核苷酸序列的基础上，根据棉铃虫多角体蛋白基因核苷酸序列，在其多态性丰富的区域设计引物（POLH1-F: 5'GTTAAGCCCGACACAATGAAGC 3'；POLH1-R: 5'ATGGGTTTGTAAAAGTTCTCCCA3'，以HaNPV的DNA为模板做PCR扩增，成功扩增出了目的片段并对PCR扩增片段进行回收、克隆和测序，测序结果表明所扩增的片段是HaNPV的多角体蛋白基因的部分序列。以纯的HaNPV的基因组为模板做PCR扩增，其检测水平可达到10fg。应用此检测体系从感染的虫卵和蛹中也成功检测到了病毒的存在。
Insect nuclear polyhedrosis virus (NPV) are invertebrate-specific pathogens that have been shown to be important in controlling populations of insects in natural epizootics and after application as biological insecticides, which play an important role in the integrated pest management (IPM) because of the virtues mentioned above.
The toxicity of Heliothis armigera nuclear polyhedrosis virus (HaNPV) to the third instar larvae was tested in the laboratory. The lethal medium dose (LD50) and concentration (LC50) are 1.99×103 polyhedron inclusion bodies(PIB) and 3.44×104PIB/ml.The lethal medium time (LT50) is varied with different infection concentrations, the more the larvae infected, the earlier they will die.
Heliothis armigera larvae are infected with concentrations of LC90、LC70、LC50、and LC30. Survivors are respectively gathered to rear in the laboratory. Mortality 、pupation ration、emergence ration and average weight of a pupa were observed in the progeny. The results are as follows: (1) Mortality in the first generation (F1) and second generation (F2) is much higher (12.22-50.42%) than that of the controls (7.50-8.33%). There is no significant distinction in larval mortality of the other generations except for treatment LC30 in the third generation (F3), whose mortality is 17.78%, significantly higher than that of control (8.83%). (2) significant distinction was observed in pupation in parent
generation (F0) and the first generation (F1) in the all treatments, the pupation ration is much lower than that of the controls, especially in the treatment LC90, whose pupation ration is only 32.14% while control 96.63%.There is no significant difference in the other generations between the treatments and the control.(3) There is significantly different in emerging ration between all the treatments and controls in parent generation (F0).Treatments in parent generation (F0), such as LC90 and LC70 ,whose emerging rate are 50.00% and 51.52% respectively, much lower than that of the control (85.99%).In the second generation (F2), only the treatment LC70(67.00%) is significantly different from the control(83.93%) while the others are no difference compared with the controls. In the other generations, no distinction was observed between treatments and controls. (4)As far as average weight of a pupa is concerned, all the treatments (0.1996-0.2356g) are significant distinction with controls (0.3036g) in the parent generation(F0). In the first generation (F1) and second generation (F2), only treatments LC70 and LC50 are significantly distinct with controls, while the others have no difference, furthermore, no distinction was observed in the other generations between all the treatments and controls.
Specific Primers were designed according to sequence of polyhedrin gene of HaNPV，polymerase chain reaction(PCR) technology was employed to detect Heliothis armigera nucleopolyhedrovirus DNA sequences from viral polyhedral inclusion bodies（PIB）.The level of sensitivity by the technology was as low as 10fg.polymerase chain reaction amplification of polyhedrin gene sequences demonstrated that the virus was present in eggs and pupae whose parents infected with HaNPV.This method may provide a better understanding of HaNPV
epizootics as well as proved the vertical transmission of the virus at molecular level.
本文对室内保存的病毒株系HaNPV的毒力进行生物测定并根据实验结果计算出LC30、LC50、LC70和LC90。用上述4种浓度感染健康的棉铃虫3龄幼虫，分别收集感染未致死的棉铃虫蛹进行传代饲养,对不同处理的各代间幼虫的死亡率、化蛹率、羽化率和平均单头蛹重等生物学特性参数进行了研究和分析，并用PCR方法对传代棉铃虫的蛹和卵进行检测，从而对NPV室内垂直传递作了比较系统和详细的研究。实验结果如下：
(1)实验室保存病毒株系HaNPV的致死中浓度（LC50）和致死中量（LD50）分别为3.44×104PIB/ml和1995PIB。当用1.0×107 PIB/ml的浓度感染棉铃虫3龄幼虫时，其感染半致死时间（LT50）为4.77天，当浓度为1.0×106PIB/ml时，LT50为5.73天，用1.0×105PIB/ml的浓度感染时为6.63天。
（2）用LC70、LC50和LC30的浓度感染寄主时，各处理的子一代（F1）和子二代（F2）的幼虫死亡率分别为15～50.42%、21.67～47.92%和12.22～34.44%，均远高于对照的7.50～8.33%；在子三代（F3）和子四代（F4）中，除了处理LC30在子三代（F3）中的幼虫死亡率（17.78%）明显高于对照（8.83%）外，其它处理与对照均无明显差异。
（3）在幼虫化蛹率方面，亲代（F0）和子一代（F1）与对照之间存在明显差异。处理LC70、LC50和LC30的幼虫化蛹率分别为61.37～72.28%、77.31～87.36%和77.39～87.05%，处理LC90亲代（F0）的化蛹率仅为32.14%，均低于对照的93.69～96.63%；在子二代（F2）、子三代（F3）和子四代（F4）中，各处理与对照均差异不明显。
（4）处理LC90、LC70、LC50和LC30的亲代和子代在羽化率方面也存在差异。
在F0代中，处理LC90和LC70的羽化率分别为50.00%和51.52%，明显低于对照的85.99%；F1代中，处理LC70的羽化率（67%）也远低于对照（83.93%），其它处理与对照差异不明显；而在F2、F3和F4代中，处理和对照差异不明显。
（5）各处理在平均单头蛹重方面与对照也有明显的差异，在亲代（F0）中，各处理（0.1996～0.2356g）与对照（0.3036g）之间差异极显著 。在子一代（F1）和子二代（F2）中，处理LC70和LC50与对照之间差异极显著，其它处理与对照无差异。各处理在子三代（F3）和子四代（F4）代中与对照无差异。
（6）在参考其它昆虫NPV多角体蛋白基因核苷酸序列的基础上，根据棉铃虫多角体蛋白基因核苷酸序列，在其多态性丰富的区域设计引物（POLH1-F: 5'GTTAAGCCCGACACAATGAAGC 3'；POLH1-R: 5'ATGGGTTTGTAAAAGTTCTCCCA3'，以HaNPV的DNA为模板做PCR扩增，成功扩增出了目的片段并对PCR扩增片段进行回收、克隆和测序，测序结果表明所扩增的片段是HaNPV的多角体蛋白基因的部分序列。以纯的HaNPV的基因组为模板做PCR扩增，其检测水平可达到10fg。应用此检测体系从感染的虫卵和蛹中也成功检测到了病毒的存在。
Insect nuclear polyhedrosis virus (NPV) are invertebrate-specific pathogens that have been shown to be important in controlling populations of insects in natural epizootics and after application as biological insecticides, which play an important role in the integrated pest management (IPM) because of the virtues mentioned above.
The toxicity of Heliothis armigera nuclear polyhedrosis virus (HaNPV) to the third instar larvae was tested in the laboratory. The lethal medium dose (LD50) and concentration (LC50) are 1.99×103 polyhedron inclusion bodies(PIB) and 3.44×104PIB/ml.The lethal medium time (LT50) is varied with different infection concentrations, the more the larvae infected, the earlier they will die.
Heliothis armigera larvae are infected with concentrations of LC90、LC70、LC50、and LC30. Survivors are respectively gathered to rear in the laboratory. Mortality 、pupation ration、emergence ration and average weight of a pupa were observed in the progeny. The results are as follows: (1) Mortality in the first generation (F1) and second generation (F2) is much higher (12.22-50.42%) than that of the controls (7.50-8.33%). There is no significant distinction in larval mortality of the other generations except for treatment LC30 in the third generation (F3), whose mortality is 17.78%, significantly higher than that of control (8.83%). (2) significant distinction was observed in pupation in parent
generation (F0) and the first generation (F1) in the all treatments, the pupation ration is much lower than that of the controls, especially in the treatment LC90, whose pupation ration is only 32.14% while control 96.63%.There is no significant difference in the other generations between the treatments and the control.(3) There is significantly different in emerging ration between all the treatments and controls in parent generation (F0).Treatments in parent generation (F0), such as LC90 and LC70 ,whose emerging rate are 50.00% and 51.52% respectively, much lower than that of the control (85.99%).In the second generation (F2), only the treatment LC70(67.00%) is significantly different from the control(83.93%) while the others are no difference compared with the controls. In the other generations, no distinction was observed between treatments and controls. (4)As far as average weight of a pupa is concerned, all the treatments (0.1996-0.2356g) are significant distinction with controls (0.3036g) in the parent generation(F0). In the first generation (F1) and second generation (F2), only treatments LC70 and LC50 are significantly distinct with controls, while the others have no difference, furthermore, no distinction was observed in the other generations between all the treatments and controls.
Specific Primers were designed according to sequence of polyhedrin gene of HaNPV，polymerase chain reaction(PCR) technology was employed to detect Heliothis armigera nucleopolyhedrovirus DNA sequences from viral polyhedral inclusion bodies（PIB）.The level of sensitivity by the technology was as low as 10fg.polymerase chain reaction amplification of polyhedrin gene sequences demonstrated that the virus was present in eggs and pupae whose parents infected with HaNPV.This method may provide a better understanding of HaNPV
epizootics as well as proved the vertical transmission of the virus at molecular level.
引文
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