Molecular Characterization of Two Mitogen-Activated Protein Kinases: p38 MAP Kinase and Ribosomal S6 Kinase From Bombyx mori (Lepidoptera: Bombycidae), and Insight Into Their Roles in Response to BmNPV Infection

Muhammad, Azharuddin; Toufeeq, Shahzad; Yu, Hai-Zhong; Wang, Jie; Zhang, Shang-Zhi; Li, Bing; Li, Zhen; Yang, Li-Ang; Hu, Pei; Ma, Yan; Xu, Jia-Ping

The purification of anti-Bmp38, anti-BmS6k serum, and preimmune serum was performed by the precipitation of ammonium sulfate according to previous method , Zhan et al. 2016 . Protein concentrations were determined by Bradford reagent (Sangon Biotech, Shanghai, China). Western blotting showed that Bmp38 and BmS6K can be recognized by anti-Bmp38 and BmS6K serum. For further analysis, the efficacy of Bmp38 and BmS6K on BmNPV, anti-Bmp38/anti-BmS6K serum (0.5 mg/ml-5 µg/larvae), and preimmune serum (0.5 mg/ml-5 µg/larvae) was administered (injection) to abdomen of the first day of fifth instar of B. mori larvae. After 30 min, 10 microliters of BmNPV (BVs) suspended in water (1 × 10 5 /ml) was administrated per os. All the experiments were performed in biological triplicates. Midgut tissues were obtained at 48 h postinfection of BmNPV. Using genomic DNA extraction kit, the isolation of genomic DNA was performed (TaKaRa). Three BmNPV GP41, IE-1, and GP64 genes were analyzed in B. mori midgut by RT-qPCR.