*3.2. Sequence Alignment and Analysis*

Using NCBI BLASTP to compare the identity of the *MKK4* gene between *P. monodon* and other species, the results showed that the *PmMKK4* gene had the highest similarity with *P. chinensis* and *P. vannamei* (Figure 2), both of which were 99.65%, which were similar to *Armadillidium vulgare*, *Ooceraea biroi*, *Harpegnathos saltator*, *Solenopsis invicta*, *Nylanderia fulva*, *Camponotus floridanus*, and *Formica exsecta*, with a similarity of 82.39%, 82.08%, 81.49%, 81.36%, 81.49%, 82.08%, and 81.36%, respectively. Using MEGA6.06 software, the phylogenetic tree of *PmMKK4* and other species was constructed based on the NJ (neighbor-joining) method, and the Bootstrap method was repeated 1000 times (Figure 3). It can be seen from Figure 3 that the *MKK4* gene of *P. monodon* had the closest genetic relationship with *P. vannamei* and *P. chinensis* and was firstly clustered into one branch.

**Figure 1.** A schematic diagram of full-length cDNA of *PmMKK4.* The black underscore indicates the initiation codon (ATG) and termination codon (TGA), the black box indicates the phosphorylation site, and the red underscore indicates the "S–I–A–K–T" kinase region.

**Figure 2.** Multiple alignment of the amino acid sequences of *MKK4* in *P. monodon* and other species. Thymosins sequence numbers used in multiple alignment included *L. vanname* (AQY45917.1), *F. chinensis* (AIY23114.1), *A. vulgare* (RXG53605.1), *O. biroi* (XP\_011345314.1), *H. saltator* (XP\_011144111.1), *S. invicta* (XP\_011160881.1), *N. fulva* (XP\_029170034.1), *C. floridanus* (XP\_011252612.1), and *F. exsecta* (XP\_029664029).

**Figure 3.** Phylogenetic analysis of *P. monodon MKK4* with other reported *MKK4*.
