Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu, China.
The Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Science, Zhenjiang, Jiangsu, China.
Arch Insect Biochem Physiol. 2022 Aug;110(4):e21896. doi: 10.1002/arch.21896. Epub 2022 Mar 30.
Pesticides are frequently used to control pests in agriculture due to their ease of use and effectiveness, but their use causes serious economic losses to sericulture when their production overlaps with agriculture. However, no suitable internal reference genes (RGs) have been reported in the study of silkworms in response to pesticides. In this study, a standard curve was established to detect the expression levels of seven RGs in different tissues of different silkworm strains after feeding with pesticides using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), including BmGAPDH, BmActin3, BmTBP, BmRPL3, Bm28sRNA, Bmα-tubulin, and BmUBC, and the stability of them was evaluated by using NormFinder, geNorm, Delta CT, BestKeeper, and RefFinder. The results showed that BmGAPDH and Bmα-tubulin were relatively stable in the midgut after feeding with fenvalerate, BmGAPDH and Bmactin3 were relatively stable in the fat body, and Bmα-tubulin and Bmactin3 were relatively stable in the hemolymph, indicating that Bmactin3 was the most suitable RG when evaluating fenvalerate, followed by BmGAPDH and Bmα-tubulin. Besides, BmGAPDH and Bmactin3 were relatively stable in the midgut after treatment with DDVP, BmGAPDH and Bmα-tubulin were relatively stable in the fat body, and BmGAPDH and Bmα-tubulin were relatively stable in the hemolymph, indicating that Bmα-tubulin was the most stable RG when evaluating DDVP, followed by BmGAPDH and Bmactin3. Of note, BmGAPDH was shared by the two pesticides. The results will be valuable for RG selection in studying the pesticide response mechanism of silkworms and other lepidopteran insects.
由于使用方便且效果显著，杀虫剂在农业中常被用于防治害虫，但当杀虫剂的使用与农业生产重叠时，会给蚕桑业造成严重的经济损失。然而，在研究杀虫剂对家蚕的作用时，尚未报道合适的内参基因（RGs）。本研究采用反转录定量实时聚合酶链反应（RT-qPCR）检测不同家蚕品系在喂食农药后不同组织中 7 个 RG（BmGAPDH、BmActin3、BmTBP、BmRPL3、Bm28sRNA、Bmα-tubulin 和 BmUBC）的表达水平，建立标准曲线，并用 NormFinder、geNorm、Delta CT、BestKeeper 和 RefFinder 评估它们的稳定性。结果表明，喂食氰戊菊酯后，BmGAPDH 和 Bmα-tubulin 在中肠中相对稳定，BmGAPDH 和 BmActin3 在脂肪体中相对稳定，Bmα-tubulin 和 BmActin3 在血液中相对稳定，表明在评价氰戊菊酯时，BmActin3 是最适合的 RG，其次是 BmGAPDH 和 Bmα-tubulin。此外，喂食敌敌畏后，BmGAPDH 和 BmActin3 在中肠中相对稳定，BmGAPDH 和 Bmα-tubulin 在脂肪体中相对稳定，BmGAPDH 和 Bmα-tubulin 在血液中相对稳定，表明在评价敌敌畏时，Bmα-tubulin 是最稳定的 RG，其次是 BmGAPDH 和 BmActin3。值得注意的是，BmGAPDH 被两种杀虫剂共享。本研究结果将有助于选择 RG 来研究家蚕和其他鳞翅目昆虫对杀虫剂的反应机制。
