中国对虾PcABCG5基因在高碳酸盐碱度胁迫下的表达模式分析

Analysis of expression patterns of the PcABCG5 gene in Penaeus chinensis under saline-alkali stress

中文关键词: 中国对虾  PcABCG5  盐碱胁迫  dsRNA

英文关键词: Penaeus chinensis  PcABCG5  Saline-alkali stress  dsRNA

中文摘要:

      为探究急性盐碱胁迫条件下中国对虾(Penaeus chinensis) ABC转运蛋白(ATP-binding cassette transporter) G5基因的表达特征，本研究应用RACE克隆技术获得中国对虾ABCG5基因，并将其命名为PcABCG5。该基因cDNA全长为2 718 bp，开放阅读框为1 923 bp，共编码640个氨基酸，预测的蛋白分子量为71.39 kDa，理论等电点为9.12，预测基因位于内质网上，为疏水性蛋白，包含1个NBD和1个TMD结构域，不包含信号肽。同源性与系统进化分析结果显示，PcABCG5转运蛋白保守性强，与斑节对虾(Penaeus monodon) ABCG5蛋白同源性最高，达98.91%。组织特异性分析结果显示，PcABCG5在各组织中均有表达，鳃中表达水平最高(P<0.05)，眼柄中最低(P<0.05)。在盐碱胁迫下，PcABCG5在鳃组织中的表达量呈先升高后下降的趋势，胁迫6 h时达到最高值(P<0.05)，为对照组的3.12倍；利用dsRNA干扰实验对该基因进行敲降，沉默PcABCG5基因会使盐碱胁迫下的对虾死亡率高达60%，比对照组高40% (P<0.05)，说明该基因的表达量越高，对中国对虾的存活越有利。本研究中受盐碱胁迫的中国对虾PcABCG5表达量高，可能是由于鳃在直接接触盐碱水环境后，通过PcABCG5介导的物质转运启动渗透压调节来应对渗透压失衡。

英文摘要:

      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.