Abstract:

Cloning and expression analysis of HMIT 

gene in Litopenaeus vannamei

LIU Hongtao, YANG Mingqiu, WANG Kun, HE Yugui
(Hainan Provincial Key Laboratory of Tropical Maricultural Technologies, 
Hainan Academy of Ocean and Fisheries Sciences, Haikou571126, China)

Abstract: The H+/myoinositol transporter (HMIT) gene, also known as solute carrier family 2 member 13 (SLC2A13) or GLUT13, is the only H+coupled transporter in the glucose transporter family. However, it does not transport glucose, but H+ coupling specifically transmembrane transports inositol. It is observed that the addition of moderate amount of inositol can improve the growth performance, nonspecific immune enzyme activity, and antioxidant capacity of Litopenaeus vannamei. Inositol is involved in glucose and lipid metabolism, osmotic pressure regulation, signal transduction, and other physiological processes in living organisms, but its absorption and transport mechanisms are largely unknown. In this study, we obtained the full sequence of HMIT gene from the hepatopancreas of L. vannamei by RACE and performed bioinformatics analysis. We used realtime fluorescence quantitative PCR to determine the distribution of HMIT expression in eight tissues, including blood cells, eye stalk, gills, heart, intestine, stomach, hepatopancreas and muscle. We also determined the expression changes of HMIT gene in each early larval developmental stage of L. vannamei, including nauplius, zoea, mysis, postlarvae, and the expression changes of HMIT in hepatopancreas and gills of L. vannamei  injected with 100 μL of decapod iridescent virus 1 (DIV1) suspension (the infection dose was about 104 viruses per g of shrimp body weight for 24 h）. The results showed that an HMIT gene was cloned from L. vannamei named LvHMIT, it was also the first report of HMIT gene in crustaceans. The open reading frame of LvHMIT was 1 650 bp in length, encoding 549 amino acids, its’ predicted molecular mass was 58 972.42 Da and the theoretical isoelectric point was 5.57. Analysis of physical properties demonstrated that LvHMIT was an unstable hydrophilic protein. A total of 70 phosphorylation sites, 2 O glycosylation sites, and 1 Nglycosylation site were predicted, some of which might affect the structure of the protein and the realization of functions such as plasma membrane localization. The absence of a typical signal peptide at the Nterminal end of LvHMIT protein was predicted. The predicted secondary and tertiary structures of LvHMIT showed that it was mainly composed of many αhelix and random coils(αhelix 43.72%; βturning 5.83%; extended chain 17.49%, and random coil 32.97%). Transmembrane structural analysis revealed the presence of 11 transmembrane helix structures in LvHMIT. The TM7 transmembrane fragment in most HMIT proteins was missing, but the secondary structure prediction at this position revealed that an αhelix that did not meet the transmembrane requirement was still present. In addition, the transmembrane helix length and the distribution of specific amino acids on the helix differed greatly between species, showing the specificity of HMIT genes in different species. Noteworthy, the predicted extracellular amino terminus of LvHMIT and intracellular carboxyl terminus, together with the absence of the above transmembrane fragment, resulted in a change in the loop structure formed between TM6 and TM7 transmembrane structures of LvHMIT (equivalent to that between TM6 and TM8 of glucose transporter proteins) from an intracellular loop to an extracellular loop, showing a difference from the typical HMIT protein structure. The predicted subcellular localization of the LvHMIT protein showed that it was mainly distributed in plasma membrane, endoplasmic reticulum, and mitochondria with percentages of 60.9%, 34.8%, and 4.3%, respectively. A typical MFS_HMIT_like domain was predicted from amino acid residue 22 to 521 in LvHMIT. Multiple sequence alignment revealed that the transmembrane structure and its arrangement in LvHMIT were relatively conserved, as well as the cysteine and glycosylation sites on the extracellular loop, including eight cysteine and CGFC motifs on the extracellular loop and the Nglycosylation site at position 410. The amino acid sequence of LvHMIT showed the highest similarity with the HMIT of Chionoecetes opilio at 69.42%. The phylogenetic tree illustrated that LvHMIT first clustered with the HMIT proteins of arthropods such as C. opilio and Hyalella azteca, and branched off from vertebrates, echinoderms, and mollusks. The tissue expression and functions performed by HMIT varied widely among species. The results of the expression distribution of LvHMIT gene in eight types of tissues using qRTPCR showed that it was most highly expressed in hepatopancreas, followed by gills, and a few expressions in heart, stomach, eyestalk, intestine, muscle and blood cells. The hepatopancreas was an important digestive and immune organ of shrimp, involved in various biological processes such as glucose metabolism and innate immunity, and gill was an important osmoregulatory organ of shrimp, equivalent to the kidney of higher animals. Therefore, LvHMIT might be involved in the biological functions of energy metabolism, immunity and osmoregulation in shrimp. Prawn larval development was metamorphic development, and it had a series of morphologically diverse larval stages, its morphology, structure, behavior, metabolism, and food habits varied with molting and metamorphosis. The result of the expression of LvHMIT in different early larval development stages of L. vannamei showed that its expression could be detected at nauplius, and was upregulated when it metamorphosed to zoea, mysis, and postlarvae, and then it was back down in the middle and late stages of zoea, mysis, and postlarvae, suggesting that it might be associated with the larval development process to some extent. DIV1 has now become a new farming disease for cultured shrimps, including Fenneropenaeus chinensis, Exopalaemon carinicauda, and Macrobrachium rosenbergii, in coastal areas of China and Thailand. The results of the expression changes of LvHMIT in hepatopancreas and gills at 24 h after the artificial infection with DIV1 showed that it was significantly downregulated (P<0.01) in hepatopancreas and upregulated (P<0.01) in gills, indicating that it was involved in the immune response process of shrimp to the virus. Inositol enhances shrimp immunity, and the response of shrimp to DIV1 infection involves inositol phosphate signaling pathways, as metabolism and glycolysis/gluconeogenesis are associated with inositol. In addition, many studies have demonstrated the involvement of HMIT genes in the regulation of phosphatidylinositol signaling pathway. In summary, this study is important for the indepth study of the structural function of HMIT gene and its role in energy metabolism, osmoregulation, larval development, and virus immunity in shrimp.
Keywords: Litopenaeus vannamei; HMIT; tissue expression; developmental expression; decapod iridescent virus 1