| 计慧君，林羿光，付彤煜，郑思春,2023,基于细菌体系生产双链RNA的条件优化[J].环境昆虫学报,(3):703-710 |
| 基于细菌体系生产双链RNA的条件优化 |
| Optimization of conditions for double stranded RNA production based on bacterial systems |
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| DOI： |
| 中文关键词: 双链RNA RNA农药生产 细菌体系 RNA提取 |
| 英文关键词:Double stranded RNA RNA pesticide production bacterial system RNA extraction |
| 基金项目:广州市民生科技攻关计划（201903010050） |
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| 摘要点击次数: 206 |
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| 中文摘要: |
| 化学杀虫剂是农业害虫防治的主要手段，然而农业害虫已对化学杀虫剂产生了抗药性。RNA农药的应用将是减少使用化学杀虫剂的一种途径。RNA农药具有专一、高效、易降解和对环境友好等优点而受到了关注，但在靶标分子、靶标分子的双链RNA（dsRNA）生产工艺和应用制剂等方面仍需进一步研究。本研究以褐飞虱Nilaparvata lugens蛋白激酶B基因的dsRNA（dsNlAKT）为例，对利用细菌体系生产dsRNA的方法进行了探究。将NlAKT构建进L4440载体后，将其转化到缺乏核酸酶（RNase Ⅲ）的大肠杆菌Escherichia coli HT115（DE3）中，进行了dsRNA生产条件的测试。结果表明：（1）当异丙基-β-D-硫代半乳糖苷（IPTG）工作浓度为0.1 mM或0.5 mM时dsRNA的产量没有明显变化，而当其工作浓度增加至1 mM时dsRNA产量明显减少；（2）在IPTG诱导时间为2~8 h范围内，添加IPTG后诱导6 h获得的dsRNA产量最多；（3）在实验室常规提取RNA方法中，增加低剂量溶菌酶预处理这一步骤可增加提取产物中dsRNA的含量。试验结果证明了采用工作浓度为0.1 mM的IPTG诱导6 h的生产条件，并在提取过程中增加溶菌酶的预处理步骤有助于获得较高的dsRNA含量。研究结果为RNA农药的生产条件提供了实验依据。 |
| 英文摘要: |
| Chemical insecticides are the main means for pest control in agriculture, however, pests have developed resistance to chemical insecticides. The application of RNA pesticides will help to reduce the use of chemical insecticides. RNA pesticides have attracted attention due to their specificity, high efficiency, easy degradation and environmental friendliness, but it still needs further studies in target genes, double stranded RNA (dsRNA) production processes and applied preparations. In this study, the dsRNA (dsNlAKT) of Nilaparvata lugens protein kinase B was used as an example to explore the conditions of dsRNA production using a bacterial system. We constructed NlAKT into the L4440 vector and transformed it into RNase III deficient Escherichia coli HT115 (DE3), and then investigated dsNlAKT production. The results showed that: (1) There was no obvious change in the yield of dsRNA when the working concentration of isopropyl-β-d-thiogalactoside (IPTG) was 0.1 mM or 0.5 mM, whereas it was clearly reduced when the working concentration was increased to 1 mM. (2) In the range of IPTG induction times of 2~8 h, the highest yield of dsRNA was obtained at 6 h. (3) The addition of a low-dose lysozyme pretreatment before the routine RNA extraction in the laboratory increased the amount of dsRNA in the extracted products. The results provide an optimal condition for IPTG induction, 0.1 mM IPTG treated for 6 h, and a pretreatment step of lysozyme for RNA extraction, which will help to obtain a higher dsNlAKT production. The experimental results provide an experimental basis for the production conditions of RNA pesticides. |
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