Analysis of Expression Patterns of PcABCG5 Gene in Penaeus chinensis Under Saline-Alkali Stress

Abstract

Penaeus chinensis is one of the most economically important species in northern China. Strong market demand requires the expansion of aquaculture production capacity of P. chinensis. Saline-alkali water covers approximately 46 million hectares in China alone, primarily distributed in the northeast, northwest, and coastal areas. Low salinity, high carbonate alkalinity, high pH, and complex ionic composition are characteristics of such waters, which cause stress to aquatic animals by interfering with physiological homeostasis. High carbonate alkalinity can directly damage the gill tissue of crustaceans. The inhabitation of ion-exchange can result in alkalosis. The understanding of response mechanisms to saline-alkali stress in P. chinensis will contribute to the sustainable development of the shrimp industry.ATP-binding cassette (ABC) transporters are one of the most prominent families of transmembrane proteins. ABC transporters can transport molecules, such as inorganic ions, sugars, amino acids, lipids, peptides, specialized metabolites, and xenobiotic agents, across membranes by binding and hydrolyzing ATP (adenosine triphosphate). Members of the ABCG subfamily consist of a single ABC cassette in the amino terminal followed by six putative transmembrane domains, and thus, are referred to as half-sized ABC transporters. Members of this family play an important role in the efflux transport of cholesterol. The ABCG subfamily participates in signal transduction, antiviral defense, and antigen presentation through hormone transport and lipid metabolism, thus helping plants adapt to changing environments. Several members of this gene family show different expression patterns in P. chinensis when exposed to saline-alkali stress. Therefore, we aimed to explore the ABC functions in resistance to alkalosis in P. chinensis.In this study, ABCG5 (GenBank accession number: OQ318160) was identified in P. chinensis and named PcABCG5. The full length of PcABCG5 was 1, 923 base pairs, encoding 640 amino acids. The estimated molecular mass was 71.39 kDa, and the theoretical isoelectric point was 9.12. Subcellular localization prediction showed that PcABCG5 was located in the endoplasmic reticulum. The PcABCG5 protein contained an NBD (nucleotide-binding domains) and a TMD domain (transmembrane domain) and had no signal peptide. Homology and phylogenetic analysis showed that PcABCG5 was highly conserved and that mature PcABCG5 shared 98.91% and 97.97% similarity with ABCG5 sequences from Penaeus monodon and Penaeus japonicus, respectively. PcABCG5 expression profiles were assessed by qPCR. PcABCG5 mRNA was detected in the eyestalk, gills, heart, muscle, intestine, hepatopancreas, stomach, and hemolymph. Our results showed that saline-alkali stress induced significant upregulation of PcABCG5 and that the expression of PcABCG5 was highest in the gills. This may be owing to the location of the gills between the external and internal environments. Osmotic pressure regulation by ABCG transporters in the gills is the primary mechanism by which P. chinensis copes with saline-alkali stress. To determine the function of PcABCG5, dsRNA against PcABCG5 was successfully injected and PcABCG5 was downregulated by 82.9% (P < 0.05). At 48 h after RNAi, we observed a 20% increase in mortality of PcABCG5 mRNA knockdown shrimp, which verified that PcABCG5 participated in the response and improved the survivability of P. chinensis under acute saline-alkali stress. Co-expressed ABCG5 and ABCG8 formed heteromeric dimers that participated in lipid transport. They also affected membrane permeability by regulating the asymmetric distribution of membrane lipids. It was also evident that saline-alkali stress induced ion and osmotic stress. We speculated that PcABCG5 may participate in the maintenance of osmotic homeostasis by hydrolyzing ATP to release energy and enhance the transport function.In conclusion, the full length cDNA of PcABCG5 was cloned in P. chinensis. PcABCG5 was upregulated under saline-alkali stress. Moreover, RNAi resulted in increased mortality of PcABCG5-silenced shrimp under saline-alkali stress. Therefore, it can be concluded that PcABCG5 is involved in the response to saline-alkali stress in P. chinenesis. Our findings provide information for further understanding the genetic basis of saline-alkali tolerance and exploring the molecular breeding of P. chinensis.

Keywords

• penaeus chinensis
• pcabcg5
• saline-alkali stress
• dsrna