*Article* **Molecular Technology for Isolation and Characterization of** *Mitogen-Activated Protein Kinase Kinase 4* **from** *Penaeus monodon***, and the Response to Bacterial Infection and Low-Salinity Challenge**

**Yundong Li 1,2,3,4, Falin Zhou 1,2, Hongdi Fan 1, Song Jiang 1, Qibin Yang 1, Jianhua Huang 1, Lishi Yang 1, Xu Chen 2, Wenwen Zhang <sup>1</sup> and Shigui Jiang 1,2,4,\***


**Abstract:** Mitogen-activated protein kinase kinase 4 (MKK4) is a component of the JNK signaling pathway and plays an important role in immunity and stress resistance. In this study, *MKK4* cDNA was cloned, and its bacterial infection and low-salinity challenge responses were researched. The full-length *PmMKK4* cDNA was 1582 bp long, with an 858-bp open reading frame (ORF) encoding a 285-amino acid (aa) protein. Results showed that *PmMKK*-4 was expressed in all examined tissues of *P. monodon.* The *PmMKK4* expression level was found to be lowest in eyestalk ganglion and highest in muscle (approximately 41.25 times than in eyestalk ganglion). Following the infection of *Staphylococcus aureus*, *PmMKK*4 was up-regulated in both hepatopancreatic and gill tissues. However, after infection with *Vibrio harveyi*, *PmMKK4* was down-regulated for a period of time in gill tissue, with fluctuating up- and down-regulation in hepatopancreas tissue. Furthermore, after infection with *Vibrio anguillarum*, gill tissue and hepatopancreas tissue showed a continuous downward trend. The *PmMKK4* gene in the gill tissue and hepatopancreas tissue of *P. monodon* was activated after low-salinity stress. The expression change of *PmMKK4* in gill tissue was more significant. The research showed that the *PmMKK4* gene plays an important role in both innate immunities after pathogen infection and adaptation in a low-salt environment.

**Keywords:** *Penaeus monodon*; *MKK4*; innate immunity; salinity stress; RNAi
