**1. Introduction**

Mitogen-activated protein kinase kinase 4 (*MKK4*) is an important regulatory kinase of the JNK signaling pathway. Activation of the JNK signaling pathway is achieved by dual phosphorylation of Thr and Tyr residues of the conserved Thr–Pro–Tyr (T–Y–P) motif [1,2]. *MKK4* can alternately activate the two pathways of JNK and p38 [3]. The first successful cloning of the *MKK4* gene was in *Xenopus laevis*, and since then, its role and molecular mechanism functions in both physiological and biochemical areas have been studied more and more deeply. However, the leading research focuses on mammals, such as humans and mice [4,5]. In studies on crustaceans, it was only found in *Daphnia pulex*, *Fenneropenaeus chinensis*, *Litopenaeus vannamei*, and *P. monodon* [6–8]. In a prior study of *P. monodon*, the expression of *MKK4* was only studied in spermatozoa and ovary developmental stages. The *MKK4* gene of *L. vannamei* shows significant changes under the stimulation of various

**Citation:** Li, Y.; Zhou, F.; Fan, H.; Jiang, S.; Yang, Q.; Huang, J.; Yang, L.; Chen, X.; Zhang, W.; Jiang, S. 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. *J. Mar. Sci. Eng.* **2022**, *10*, 1642. https://doi.org/ 10.3390/jmse10111642

Academic Editor: Ka Hou Chu

Received: 29 September 2022 Accepted: 1 November 2022 Published: 3 November 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

bacteria and is activated by the phosphorylation of upstream genes and then activated JNK, which plays an important role in the antibacterial response [3,9]. The *MKK4* gene of *Ctenopharyngodon idella* is involved in the immune stress response to muramyl dipeptide in the intestinal tissue of *C. idella* [10].

In order to further improve the molecular function study of the *MKK4* gene in *P. monodon*, this study cloned the entire length of the *MKK4* gene in *P. monodon*, analyzed the expression of *MKK4* in various tissues of *P. monodon*, and explored the effect of different degrees of pathogen stimulation and salinity stress. It is necessary to explore the molecular function and role of *MKK4* in low-salinity stress in *P. monodon* to provide more basic data to determine the molecular mechanism of the response to salinity stress in *P. monodon*.

#### **2. Materials and Methods**

#### *2.1. Experimental Animals and PmMKK4 cDNA Cloning*

*P. monodon* specimens, each with a body length of 7–10 cm and a body weight of 8–12 g, were selected and cultured for 1 week in a 25–28 ◦C seawater environment with full aeration at the same time. Based on the unpublished transcriptome of *P. monodon* tissues constructed in our laboratory, specific primers, namely, *PmMKK4*-F1, *PmPKK4*-R1, *PmMKK4*-F2, and *PmPKK4*-R2 (Table 1), were designed by Primer Premier 5.0 (RuiBiotech, Guangzhou, China). The 5 and 3 ends of *PmMKK4* were acquired by using the rapid amplification of the cDNA end (RACE) method (Clonetech, Tokyo, Japan). Through the 5 or 3 RACE-PCR, PCR was performed initially with *PmMKK4*-5G1 (*PmMKK4*-3G1 for 3 ) and universal primers UPM Long and UPM Short, followed by semi-nested PCR with *PmMKK4*-5G2 (*PmMKK4*-3G2 for 3 ) and a nested universal primer NUP (Table 1). The PCR conditions were designed as follows: one cycle of 94 ◦C for 3 min, 35 cycles of 94 ◦C for 30 s, 67 ◦C for 30 s, and 72 ◦C for 45 s, followed by a final cycle of 72 ◦C for 10 min. The PCR products were then gel-purified and sequenced, and the sequences were determined after the analysis.


**Table 1.** Sequences of primers used in this study.

#### *2.2. Bioinformatic Analysis*

The ORF Finder (http://www.ncbi.nlm.nih.gov/orffinder/, accessed on 10 May 2021) was used to predict the open reading frame. The transeq in EMBOSS (http://www. bioinformatics.nl/emboss-explorer/, accessed on 10 May 2021) was utilized to translate its amino acid sequence. The predicted amino acid sequence was compared with the protein database by using the BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 20 May 2021) tool in NCBI. Multiple sequence alignments were performed using Clustal X V1.83 software. ExPASy ProtParam (https://web.expasy.org/protparam/, accessed on 20 May 2021) was used to predict the isoelectric point and the theoretical molecular mass. Protein domain analysis was performed using SMART 4.0 (http://smart.embl-heidelberg. de/smart/set\_mode.cgi?GENOMIC=1, accessed on 20 May 2021). Glycosylation sites were predicted using the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/, accessed on 20 May 2021). Phosphorylation sites were predicted using the NetPhos 3.1 Server (http://www.cbs.dtu.dk/services/NetPhos/, accessed on 20 May 2021). The phylogenetic tree was constructed based on the maximum likelihood method using Clustal X and MEGA 6.0 software.
