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

Real-time quantitative polymerase chain reaction (RT-qPCR) was performed for the examination of expression level of Bmp38 and BmS6K in different tissues and under different post-treatments. The primers utilized in this study were designed by Primer Premier 5.0 (www.premierbiosoft.com; Table 1 ). According to the instructions of manufacturer, the real-time quantitative PCR were set with TB Green premix ex Taq (TaKaRa). Briefly, in a 25-µl reaction mixture, the RT-qPCR was performed containing 12.5-µl TB Green, 9.5-µl double-distilled water, 1-µl forward primer, 1-µl reverse primer, and 1-µl cDNA template. The thermal cycling program was set as follows: denaturation at temperature of 95°C for time period of 30 s, 40 cycles of temperature 95°C for 5 s, and 72°C for time of 20 s. Using the 96-well plates with multicolor RT-PCR detection system, the reactions were carried out (Bio-Rad). 2 −ΔΔCt. method was adopted according to the previously published protocol for the calculation of relative expression level (Yu et al. 2007 ). There were three biological sample replications. The B. mori GAPDH was used for normalization. ANOVA and LSD a posteriori tests were used for statistical analysis in SPSS software (P < 0.05).