*2.3. RNA Extraction and Sequencing*

Using the HiPure Fibrous RNA Plus Kit (Megan, Guangzhou, China), total RNA from ovarian tissue was extracted. The RNA products were checked for integrity using 1.2% agarose gel electrophoresis, and concentration and purity were checked using Nanodrop (Thermo Scientific NC2000, Waltham, MA, USA). Finally, the concentration was ≥1.2 μg/μL, the total amount was ≥60 μg as the sample quality standard, and the samples that met the requirements were used in the experiments.

#### *2.4. PmNHE cDNA Cloning*

We created the specific primers NHE-F and NHE-R (Table 1) based on Primer Premier 5.0 (RuiBiotech, Beijing, China). The 5 and 3 ends of *PmNHE* were obtained using a method developed by Clonetech in Japan. The following conditions were used in the PCR: 1 cycle at 94 ◦C for three minutes, 35 cycles at 94 ◦C for 30 s, 67 ◦C for 30 s, and 72 ◦C for 45 s, and a final cycle at 72 ◦C for ten minutes. After being sequenced and purified on a gel, the PCR products were analyzed. The product was purified according to the operating instructions of the purification kit (TIANGEN, Guangzhou, China).



#### *2.5. Bioinformatic Analysis*

Using ExPASy ProtParam software, protein physicochemical properties were predicted (http://web.expasy.org/protparam/, accessed on 5 October 2021), and domain analysis was conducted using SMART4.0 (http://smart.embl-heidelberg.de/, accessed on 5 October 2021). NetPhos 2.0 (http://www.cbs.dtu.dk/services/NetPhos/, accessed on 5 October 2021) and NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/, accessed on 5 October 2021) were used for predicting protein phosphorylation and glycosylation, respectively. The Clustal X software was used to align multiple sequences, and then, BioEdit and MEGA 6.0 software was used to construct phylogenetic trees.

Combining cDNA and RACE-PCR sequences was used to create the full-length *PmNHE* gene using DNA-man software version 10. The open reading frame (ORF) was located using ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 8 October 2021). EMBOSS (http://www.bioinformatics.nl/emboss-explorer/, accessed on 8 October 2021) was used to predict the amino acid (aa) sequence. The protein physicochemical properties and aa sequences were predicted using the ExPASy ProtParam software (http://web.expasy.org/protparam/, accessed on 8 October 2021). The SMART4.0 online program was used to analyze protein domains (http://smart.embl-heidelberg.de/, accessed on 10 October 2021). The protein phosphorylation sites were predicted using the NetPhos2.0 program (http://www.cbs.dtu.dk/services/NetPhos/, accessed on 10 October 2021), and the protein glycosylation sites were predicted using the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/, accessed on 10 October 2021). The Clustal X was used to align multiple sequences, and BioEdit and MEGA 6.0 were used to create a phylogenetic tree.

#### *2.6. qRT-PCR Analysis of PmNHE mRNA Expression*

The expression of *PmNHE* mRNA in various organs was discovered using qRT-PCR. As the reference gene, elongation factor 1α (EF1a) was utilized [16,17] (Table 1). *PmNHE* is compatible with the reaction condition for EF1a. The Roche Light Cycler® 480II was used to perform qRT-PCR with green fluorescence measurement. The relative CT method (2−ΔΔCT) was used to obtain the PCR data [13,15,16]. For each time point in the experiment, three individuals were sampled, and for RT-PCR, three technical replicates were made to ensure accuracy. A one-way ANOVA and Tukey's multiple range test were used for the statistical study (IBM, New York, USA). At *p* < 0.05, the differences were deemed to be significant. Data from the tests are displayed as mean SD (standard deviation).

#### *2.7. RNA Interference*

Using Primer Premier 5.0, the primers dsNHE-f, dsNHE-r, dsGFP-F, and dsGFP-R (Table 1) required for the synthesis of dsNHE were created. Ex Taq was used as a template to amplify the T7 promoter-containing DNA fragment. Clear and bright bands were obtained, agarose gel recovery was performed according to the instructions of the gel recovery kit, and the cDNA was stored at −20 ◦C with a concentration greater than 125 ng/μL and meeting the experimental requirements for later use. The synthesis of dsRNA was carried out according to the instructions of the T7 RiboMAXTMExpress RNAi System kit. Purification was performed to reserve the obtained dsRNA. Using the same procedure, green fluorescent protein (GFP) double-chain RNA and the pDGFP recombinant vector were produced.

In this experiment, dsRNA was injected at a ratio of 35 μg/g into *P. monodon* at a weight of (5.0 ± 1.0) g. There were three groups of injection tests: groups that received injections of PBS, dsGFP, and ds*PmNHE*. Before the injection, healthy shrimp were randomly taken from the molting interphase. Their intestines were set in RNAlater as 0 h samples for inspecting RNAi efficiency. The shrimp specimens were transferred to a bucket containing high-concentration ammonia nitrogen 24 h after being injected. Every three hours, dead shrimp from each group were recorded and collected.

Healthy shrimp were taken from each treatment group at 3, 6, 9, 12, 24, and 48 h following exposure to high-concentration ammonia nitrogen stress, and their intestines were collected and set in RNAlater. Ten shrimp were simultaneously injected with dsNHE and dsGFP and set in two separate buckets. In order to evaluate the efficacy of dsRNA interference, intestinal tissues were randomly collected after 24 and 48 h and set in RNAlater as samples. All of the RNA samples were mixed with RNAlater and kept at −80 ◦C.

#### **3. Results**

#### *3.1. PmNHE Sequence Display and Bioinformatics Analysis*

The full-length *PmNHE* cDNA was obtained by splicing the open reading frame and 5 /3 non-coding region sequence sequencing results. *PmNHE* was 2788 bp long (GenBank accession No. MT164534), including an ORF of 2643 bp, a 5 -untranslated region (UTR) of 76 bp, and a 3 UTR of 78 bp (Figure 1). The ORF of the *PmNHE* gene encoded 877 amino acids with a molecular weight of 97.97 KDa and a theoretical isoelectric point of 6.54. Bioinformatics prediction analysis showed that *PmNHE* had a variety of functional sites, contained 23 phosphorylation sites (20 serine sites and 3 threonine sites), 6 glycosylation sites (highlighted in green font), and 12 transmembrane domains (highlighted with light blue line segments). The sequence contains a sodium/proton *exchanger* domain (highlighted by gray shading) at 78-490 aa. The poly A structure is used in the 3 -UTR sequence (highlighted in italics).

The *PmNHE* (Genebank No. MT164534) start code (ATG) and termination code (TAA) are listed in a square box. The signal peptide sequence is highlighted in red font. The glycosylation site is highlighted in green font, and the *Na+/H+-exchanger* domain is expressed in gray. The Poly A structure is highlighted in italics.

#### *3.2. Phylogenetic Tree Analysis and Multiple Sequence Alignment*

In NCBI, the amino acid sequence of the *NHE* gene of *P. monodon* was compared with other species, and it was found that it had a high homology with the amino acid sequence of the NHE protein of *Penaeus vannamei*.

The NJ evolutionary tree of *PmNHE* and this protein of other species was constructed by MEGA6.0 (Figure 2). The results show that the relationship between invertebrates was the closest, and the relationship between *Penaeus vannamei* and *P. monodon* was the closest and clustered together, and they clustered into a branch with *Scylla olivacea* and *Hyalella azteca*.

**Figure 1.** Nucleotide and deduced amino acid sequence of *PmNHE*. (\* *termination codon, tga*).

**Figure 2.** Phylogenetic analysis of *PmNHE. (*represents the *PmNHE* gene obtained in this study).

The results of amino acid sequence multiple alignment analysis (Figure 3) show that the amino acids of *P. monodon* NHE and other species have high homology, among which, *PmNHE* and *Penaeus vannamei* (qcr98514.1) have the highest similarity of 93.92%; the protein similarity with *Limulus polyphemus* (xp\_013784312.1) was 42.71%; the protein similarity with *Portunus trituberculatus* (anv19765.1) was 35.77%.
