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

Total genomic DNA was isolated using midgut of P50 and BC9 infected with BmNPV injection and oral inoculation, and using the genomic DNA extraction kit, the noninfected larvae were isolated at time 6, 12, 24, 48 and 72 hpi (Dalian, Takara, China) . To measure the quantity of all DNA samples, a spectrophotometer (Nano-Drop 2000; Thermo Fisher Scientific, New York, NY) was used. To check the purity of all DNA samples, A 260/280 and A 260/230 absorbance ratio was used, and 1.0% agarose gel electrophoresis was used to confirm the integrity of DNA. The GP41 primer was showed in Table 1 . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; GenBank ABA.43638) gene was used as a reference gene because of its suitability described previously (Guo et al. 2016) . The real-time qPCR was performed in a 25-µl reaction mixture containing 12.5 µl of TB Green premix EX Taq (TaKaRa). PCR amplification was performed in triplicate wells. The thermal cycling program was set as denaturation at 95°C for 30 s and 40 cycles of 95°C for 5 s, 60°C for 30 s, and 72°C for 20 s. All these reactions were carried out in 96-well plates with a Multicolour Real-Time PCR Detection System (Bio-Rad, Hercules, CA). 2 −ΔΔCt method was used to calculate the relative expression level followed the previously published protocol (Yu et al. 2007 ). Analysis of variance (ANOVA) and least significance difference (LSD) a posteriori tests were used for statistical analysis by using SPSS software (P < 0.01).