The role of 14-3-3 genes from black tiger shrimp Penaeus monodon on osmotic regulation

Abstract

The cDNAs encoding two different isoforms of 14-3-4 protein, namely 14-3-3A and 14-3-3B, were identified from the Penaeus monodon EST database. Sequence analysis revealed a complete coding sequence (CDS) of 14-3-3A while a partial sequence of 14-3-3B was obtained. In this study, we attempt to isolate a complete CDS of 14-3-3B cDNA using 5' Rapid Amplification of cDNA Ends (5' RACE) and reverse transcription (RT)- PCR. However, the complete CDS of 14-3-3B was not successfully isolated although several conditions were applied. This trouble was probably due to a strong secondary structure at the 5' end of 14-3-3B mRNA. A phylogenetic analysis of 14-3-3 sequences suggested that P. monodon 14-3-3A was closely related to 14-3-3 Zeta isotype whereas the 14-3-3B was more closely related to 14-3-3 Epsilon isotype. The effect of salinity on mRNA expression of the two isoforms of P. monodon 14-3-3 was determined in shrimp gill using a semi-quantitative RT-PCR. It was found that shrimps reared under low salinity (3ppt) showed higher expression of 14-3-3B mRNA than those acclimatized at high salinity (25 and 40 ppt). A significant decrease in the mRNA level of 14-3-3B was observed in shrimp transferred from 3 ppt to 40 and 25 ppt while the mRNA level of 14-3-3A remained unchanged or slightly changed over the salinity range tested. The results suggest that 14-3-3B probably plays a role in hypo-osmotic regulation in P. monodon. Interestingly, it was found that not all shrimp samples collected from different farms, expressed 14-3-3B isoform but shrimps that expressed 14-3-3B were more tolerant to sudden salinity transfer than those that did not. The cumulative mortalities of shrimps expressing 14-3-3B from two shrimp groups from farm I (Tamai district) was about 23 % and 57 % in day4 after low salinity stress while those of shrimps not expressing the gene in the two groups from farm II (Lamsing district) were 100 %. In addition, knockdown of 14-3-3B gene by RNA interference (RNAi) resulted in a significant decrease in total ATPase activity in shrimp gill suggested that 14-3-3B might be involved in the regulation of ATPase.