Molecular characterization, expression analysis of 14-3-3 beta/alpha and the effect of RNA interference on ion transporter protein Na+-K+-ATPase, Na+-H+-exchanger and CFTR in turbot (Scophthalmus maximus).

Abstract

To understand the role of 14-3-3 beta/alpha in hypoosmotic regulation of turbot (Scophthalmus maximus), we characterized the 14-3-3 beta/alpha gene and analyzed the tissue distribution and its gene transcriptional patterns in the main expressed tissues under low salt stress. The 14-3-3 beta/alpha cDNA is 892bp in length, incorporating an ORF of 774bp with a putative primary structure of 257 residues. The deduced amino acid sequences shared highly conserved structures with other eukaryotes. Quantitative real-time PCR results showed that the 14-3-3 beta/alpha transcripts were widely expressed in various tissues of turbot, with most abundant in the gill (P<.05), to a lesser extent in the kidney, intestine, brain and spleen, and at low levels in the pituitary and other tissues examined. And the expression of turbot 14-3-3 beta/alpha exhibited a trend of increasing first and then decreasing with the time of stress under low salt stress, and the highest value appeared in 12h (P<.05). After injecting different concentrations of dsRNA, the mRNA expression of 14-3-3 gene decreased significantly during the monitoring period, and the best interference effect was achieved 12h after injecting 4μg/g dsRNA. For the first time, the gene was silenced in fish by intramuscular injection of dsRNA. It also provides a new and effective way to study gene function at the individual level. Moreover, the mRNA interference of 14-3-3 beta/alpha would cause changes in the expression of several ion channel proteins, for example, the decrease of Na+-K+-ATPase and Na+-H+-exchanger and the increase of CFTR. As a result, 14-3-3 beta/alpha appears to be an important molecular regulator for osmosensory signal transduction in gill of turbot.