Aquaporins are transmembrane proteins that allow the passage of water and small uncharged solutes, such as glycerol across lipid bilayers (Denker et al., 1988; Sui et al., 2001; Agre, 2004; Walz et al., 2009). Thus far, 13 members of mammalian aquaporins (AQPs) have been identified (AQP0-12), and most of them have been well characterized. In general terminology, the AQP family can be divided into three major subgroups based on their permeability characteristics， including the classical water-selective aquaporins (AQP0, -1, -2, -4, -5, -6, -8); the glycerol transporting aquaporins, known as aquaglyceroporins (AQP3, -7, -9, -10); and the unorthodox aquaporins (AQP -11 and -12) whose function is still being elucidated (Agre, 2006; Cerda and Finn, 2010; Soto et al., 2012; Abascal et al., 2014; Finn et al., 2014). Studies have shown that AQP plays an important role in the transport of ions, water molecules, and osmotic pressure adjustment (Robinson et al, 1996; Martinez et al, 2005; Holm et al, 2005; Wang et al, 2014). As the osmotic pressure of the body changes, the water transport pattern changes accordingly (Tan et al, 2018). This not only directly affects the expression of aquaporin, but also affects the synthesis of aquaporin. It is performed in the cell fluid, therefore, AQP plays an important role in maintaining the stability of osmotic pressure inside and outside the cell (Sade et al., 2010). By changing the size of its pore size and spatial structure, aquaporin makes the channel hydrophilic and selective permeable, limiting the passage of most ions, allowing only water molecules and specific ions to pass through. This special structure and function of aquaporin ensures that the osmotic pressure of the body always maintains a dynamic balance (Tipsmark et al., 2010; Yakata et al., 2011). At present, research on aquaporins is mainly concentrated in plants, mammals and fish (Cutler et al., 2002; Uehlein et al., 2006; Tipsmark et al., 2010; Giffard-Mena et al., 2011; Guo et al., 2017; Vorontsova et al., 2018), and research in crustaceans is scarce (Chung et al., 2012; Wang et al., 2014; Foguesatto et al., 2017).

Eriochir sinensis is widely distributed in rivers and lakes in the middle and lower reaches of the Yangtze River in China and is one of the important commercial crab species in China (Cheng et al., 2000). Studies have shown that during the growth and development of Eriocheir sinensis, molting is the most important part. Small changes in the process of molting will cause death. The success rate of molting is related to the success or failure of Eriocheir sinensis breeding. However, the current research on molting mainly focuses on enzyme activity, immunity, ecdysone and energy (Cheng et al., 2000; He et al., 2016; Huang et al., 2018), while the gene of aquaporin, which plays an important role in water transport, has not been reported.

Through molecular cloning technology, the full-length cDNA sequence of AQP11 gene was obtained, and the expression level of AQP11 gene in muscle and intestinal tissue of Eriocheir sinensis during molting was studied, in order to further reveal the role in the process of Eriocheir sinensis molting process provides a theoretical basis.

1 Results

1.1 cDNA cloning, sequence analysis of AQP11

The complete cDNA sequence of AQP11 was 1 746 bp in length with an open reading frame (ORF) of 807bp corresponding to 269 amino acids, and a 476bp 3’-untranslated region (UTR) and a 463bp 5’-UTR. The calculated molecular weight (MW) and isoelectric point (PI) of the deduced aquaporin are 29.46kDa and 5.38, Isoleucine (Leu) and Valine (Ala) have the highest content, accounting for 11.2% and 10.1%, respectively. Amino acid composition analysis showed that the AQP11 protein has 23 negative amino acid residues (Asp and Glu), 17 positively charged amino acid residues (Arg and Lys), The atomic composition is C₁₃₅₂H₂₀₈₈N₃₃₂O₃₆₈S₁₈, and the instability coefficient is 28.28, which proves that the protein is classified as stable. The aliphatic amino acid index is 106.98, indicating that the protein has high heat resistance. TMHMM 2.0 software analysis showed that AQP11 contains six transmembrane domains (located at positions 62-84, 159-181, 194-216, and 231-250, respectively), and two NPV (Asn -Pro-Val) domains. Signalp 5.0 software was used to detect the absence of a signal peptide at the N-terminus of the Chinese mitten crab AQP11 gene, which is a non-secreted protein (Figure 1).

1.2 AQP11 gene homology analysis and evolutionary tree construction

Amino acid sequence of AQP1 and homologs from other crustaceans were aligned using BLAST, and functional domains are highly conserved (Figure 2). Pair-wise comparison of the Eriochir sinensis AQP1with other AQP1 from different species was divided Litopenaeus vannamei (XP_027231404.1, 82% identity), Athalia rosae (XP_012267381.1, 48% identity), Cephus cinctus (XP_015595607.1, 49% identity), Trinochestia longiramus (KAF2368393.1, 70% identity), Spodoptera litura (XP_022822820.1, 48% identity), Zootermopsis nevadensis (XP_021940669.1), respectively. Comparing with other known AQP11 sequences from crustaceans revealed that the AQP11 amino acid sequence has all the typical conserved features of members of the AQP11 family, including four transmembrane domains, two NPV (Asn-Pro-Val) domains and two conserved sequences TACPY, YSGGYSNP (Figure 2).

The phylogenetic tree of AQP11 amino acid sequences was constructed to clarify the evolutionary relationship between AQP11 and the AQP11 molecules of other species. The resulting phylogenetic tree also showed that the AQP11 sequences fall into distinct lineages. The sequence of AQP11 was clustered with sequences of AQP11from the other crustaceans firstly, then clustered with other AQP11 sequences from invertebrates and vertebrates (Figure 3).

1.3 Tissue distribution of AQP11 mRNA expression

Real-time fluorescence quantitative PCR was used to analyze the expression and distribution characteristics of Eriocheir sinensis AQP11 gene in different tissues. The results showed that AQP11 gene was expressed in all tissues. Among them, the highest expression in the intestine, followed by brain, muscle and thoracic ganglion, the lowest expression in the hepatopancreas, gills and blood cells (P <0.05, Figure 4).

1.4 Expression analysis of AQP11 gene during molting cycle

The expression of AQP11 in the intestine during the molting cycle of Eriocheir sinensis is shown in Figure 5. The expression level of AQP11 was at a low level between the intemolt stage (C stage) and the premolt stage (D stage), but significantly increased and reached the highest during the ecdysis stage (E stage) (P <0.05, Figure 5), and postmolt stage (AB stage) The expression level remains unchanged, and it is still significantly higher than that between the intermolt stage (C phase) and the premolt stage (D stage) (P <0.05, Figure 6).

Real-time PCR detected the expression of AQP11 gene in the molting cycle of Eriocheir sinensis muscle. The expression level in the intemolt stage (C stage) was at a low level, but it increased significantly (P <0.05) in the premolt stage (D stage), and reached the highest level in the ecdysis stage (E stage) (P <0.05), and then The expression level decreased at the postmolt stage (AB stage) (P <0.05, Figure 7).

2 Discussion

In this study, we isolated the full-length AQP11 gene from the intestine of Eriocheir sinensis and studied the tissue expression distribution and the effect of molting on its expression. The gene has a total length of 1746bp, contains an open reading frame of 807bp and encodes 268 amino acids. Protein structure analysis found that the protein encoded by AQP11 gene contains 4 transmembrane regions and two NPV (Asn-Pro-Val) structural units. In mammals (AQP0-AQP10), the water transport structural unit is NPA (Asn-Pro-Ala), while the water transport structural units of AQP11 and AQP12 genes are variable (Yeung and Cooper, 2010; Yakata et al., 2007 ), Similar to the results of this experiment, there is also a variation in the water delivery unit in the AQP11 gene of Eriocheir sinensis, that is, valine (Val) replaces alanine (Ala). Although this change reduces the transport speed of AQP11 to water, it ensures long-term uniform transport of water to AQP11, enhances the function of AQP11 in ion transport, and expands tissue distribution range (Yakata et al., 2011; Bestetti et al., 2020). Phylogenetic tree analysis showed that Chinese mitten crab AQP11 and Litopenaeus vannamei AQP11 are a branch, and then they are a branch of Drosophila ananassae, Spodoptera litura and Zootermopsis nevadensis.

The Eriocheir sinensis AQP11 gene is expressed in gills, hepatopancreas, muscle, intestine, stomach, heart, brain ganglion and thoracic ganglion, with the highest expression in the intestine, followed by cerebral ganglion, in hepatopancreas , Gill and heart expression levels are low. AQP11 is expressed in mammalian tissues and organs (Kenichi et al., 2009). When interfered with the expression of the AQP11 gene in the kidneys of mice, it was found that swelling and cavitation of the proximal tubules of the mouse kidneys within a few hours after the interference led to a reduction in the rate of water reabsorption of the mice and death of the mice. This further proves the effect of AQP11 gene on water transport (Morishita et al., 2005).

Molting is the most important link in the growth and development of crustaceans. After the crustacean (exoskeleton) is hardened, it will form a fixed space and prevent the tissue from growing like its vertebrate. Every once in a while crustaceans must shed old exoskeletons, grow new ones, and complete growth (Chen, 2006). The study found that during the molting cycle of shrimps, aquaporins ensure the smooth progress of molting by adjusting the cell size. In the premoult (D stage), the amount of water in the cell is reduced to reduce the volume of tissue to achieve the purpose of smooth molting. In the ecdysis stage (E stage), the cell volume is expanded to reach the maximum size of the tissue before the outer case hardens. In the postmolt stage (AB stage), the excess water in the cells is eliminated to provide space for the growth and development of the body. When the osmotic pressure inside and outside the cell is again balanced, part of the aquaporin activity is restored to maintain the normal physiological metabolism of the body (Foguesatto et al., 2017; Huang et al., 2018; Chen, 2016). This is similar to the results of this experiment. This adjustment method improves the success rate of molting of Eriocheir sinensis. But its specific regulation mechanism still needs further study.

3 Materials and Methods

3.1 Animals and Reagents

Healthy Chinese mitten crabs (Eriocheir sinensis) were taken from Shanghai Ocean University (Shanghai Chongming Breeding Base) and weighed (5.82 ± 2) g. Temporarily reared in an indoor circulating aquaculture system, two floors above and below, 6 breeding ponds, temporarily reared for 1 week, breeding water temperature 26 °C ± 2 °C Feed commercial feed regularly at 18:00 every day and suck out the bait from yesterday. The molting cycle was classified by microscope observation. The crabs in the same period were concentrated for monoculture, and the experiment was started after the first unified molting (Kang et al., 2013).

Trizol Reagent, PrimeScript ™ II 1st Strand cDNA Synthesis Kit, SMARTer RACE 5 '/ 3' Kit, pMD ™ 19-T Vector Cloning Kit and PrimeScript ™ RT Master Mix (Perfect Real Time) Both were purchased from Dalian Bao Biological Engineering (TaKaRa) Co., Ltd; DNA gel recovery kits and TOP10 competent cells were purchased from TIANGEN BIOTECH (BEIJING) Co., Ltd; ChamQ Universal SYBR qPCR Master Mix was purchased from Nanjing Nuoweizan Biological Co., Ltd.

3.2 Sample collection

Observe the changes in the tip of the third jaw foot of the Eriocheir sinensis by microscope, and divide the Eriocheir sinensis at the same molting stage into one group (AB stage, C stage, D stage, E stage) (Kang et al., 2013) After its uniform molting, the experiment was started, and the experimental crabs were placed in a monoculture circulating water tank, with 8 parallels in each group and 20 parallels in each group. Muscles and intestines were taken from each group, and cryopreserved in liquid nitrogen for RNA extraction. Eight animals were taken from each molting stage. 6 gills, hepatopancreas, muscles, thoracic ganglia, intestines, stomach, brain, and healthy nitrogen of Eriocheir sinensis, which were healthy during the molting period (C stage), were taken for liquid RNA extraction.

3.3 Extraction of total RNA and synthesis of the first strand of cDNA

The total RNA of intestine, muscle and gill tissue was extracted with Trizol reagent, and the quality and integrity of RNA were detected using 1.0% agarose gel electrophoresis; the RNA concentration was determined using nucleic acid quantifier. Take equal amounts of RNA and mix evenly. Follow the instructions on the PrimeScript ™ II 1st Strand cDNA Synthesis Kit to synthesize the first strand cDNA.

3.4 Cloning and identification of AQP11 cDNA

Based on the obtained core fragments, 5'RACE and 3'RACE specific primers were designed, and the 5 'and 3' ends were rapidly amplified using SMARTer RACE 5 '/ 3' Kit. The required primers and sequencing are completed by Biotechnology (Shanghai) Co., Ltd. After the 3'RACE and 5'RACE amplification products were detected by gel electrophoresis, the target fragments were recovered for ligation transformation and PCR amplification detection, which was sent to the company for detection as expected (Table 1).

3.5 Sequence analysis

Use DNAMAN software to sequence the measured sequences, and then use NCBI ORF find (http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi) online software to predict the open reading frame of AQP11 gene. At the same time, NCBI BLAST online software was used to perform homology alignment between the nucleotide sequence of the AQP11 gene and the deduced amino acid sequence. Use ExPASy-SIT tool to predict amino acid functional domains, ExPASy-ProtParam tool online software to predict protein physicochemical properties, use SWIS-MODEL (http://swissmodel.expasy.org/interactive) and TMHMM 2.0 (http: // www. cbs.dtu.dk/services/TMHMM/) online software for predictive analysis of protein three-dimensional structure and transmembrane region. DNAMAN software was used to compare with amino acid sequences of other species, and MEGA 5.0 software was used to construct the phylogenetic tree.

3.6 Quantitative real-time PCR (qRT- PCR) analysis of AQP11 expression

Expression of AQP11 mRNA in different tissues was analysed by qRT-PCR. PrimeScriptTM RT Master Mix (Perfect Time) was used to reverse transcribe the total RNA from each tissue of Eriocheir sinensis to synthesize cDNA. Real-time specific primers for AQP11 and 18S were designed (Table 1). The 10 μl qRT-PCR reactions contained 5 μl ChamQ Universal SYBR qPCR Master Mix (Vazyme), 3.6 μl PCR-grade water, 1.0 μl diluted cDNA template (two-fold diluted), and 0.2 μl (10 μM)of each forward and reverse primer. qRT-PCR was performed in an ABI PRISM® 7500 Sequence Detection System (Applide Biosystems, USA) according to the manufacturer’s protocol at 95 °C for 30 s, followed by 40 cycles at 95°C for 5 s, and 60 °C for 34 s. This was followed by melting curve analysis to confirm single PCR products. Each sample was run in triplicate. AQP11 expression levels were calculated by the 2^−ΔΔCt comparative CT method.

Authors’ contribution

ZL is responsible for biological information analysis, real-time fluorescence quantitative PCR experiments and article writing; Pang Yangyang is responsible for experimental sampling and article modification; Yang Hang is responsible for the revision and proofreading of the paper; Yang Zhigang is the designer and person in charge of the project. All authors read and agreed to the final text. 

Acknowledgements

This study was financially supported by the National Key Research and Development Program of China (No. 2018YFD0900603), the China Agriculture Research System-48, and the capacity promoting Project of Shanghai Engineering and Technology Center from Shanghai Municipal Science and Technology Commission (No. 19DZ2284300).

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