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

Silkworms were dissected for the collection of the midgut, hemolymph, fat body, integument, head, and silk gland (pathogen challenged) and using TRIzol reagent (Invitrogen, Carlsbad, CA) as per manufacturer's protocol. A Nano-Drop 2000 Spectrophotometer (Thermo Fisher Scientific) was used to determine the concentration, and purification of RNA samples was noticed at A 260/280 . 1% agarose gel electrophoresis was used for checking RNA integrity. According to previous protocol, the RNA samples were then reverse transcribed into cDNA by using Primescript RT Kit with gDNA Eraser (Takara; Yu et al. 2018) . Briefly, the concentration was adjusted at 1 µg/µl with nuclear-free water for each RNA sample, and in a 20-µl reaction mixture, the total RNA was reversely transcribed. The mixture was kept in an incubator for 15 min at 37°C and then kept for 5 s at 85°C. The cDNA was kept at temperature of −20°C for future use.