A NOVEL shRNA TOOL TO STUDY TARGETED SILENCING EFFICIENCY OF IRAK4 GENE IN Penaeus monodon

Gene silencing by RNA interference (RNAi) is a widely used approach for target-specific knockdown of gene expression. RNAi has become a powerful research tool in gene function studies nowadays. The basic aim in this study was to construct a novel shRNA construct which is named pSh-IRAK4 for the purpose of studying IRAK4 gene silencing effect and its response generated on the immune mechanism of Penaeus monodon.

The target region of IRAK4 gene was selected and its sense and antisense strands were synthesized and were cloned in pSUPER vector (Fig.1). Its transcript can form a short hairpin RNA (shRNA) with inverted, repeated sequence separated by a short loop sequence. The shRNA then was processed into functional siRNA to degrade target IRAK4 mRNA and silence its expression. This recombinant construct was identified by enzyme digestion (Fig.2) and DNA sequencing.

The recombinant vector inhibited transcription of the IRAK4 gene. Quantitative real-time PCR analysis revealed that pSh-IRAK4 construct suppressed IRAK4 mRNA production to more than 80% of that in the control cells (Fig.3). Selection of an effective target sequence is the key point of RNA interference. On the basis of these results, we selected IRAK4 which is an essential factor for TLR-mediated activation of the host's immune functions subsequent to the pathogen contact.

Our result support that sequences designed and cloned efficiently into pSUPER vector could be used to reduce the expression of the target gene. Thus, this study could be projected to set benchmark on role of IRAK4 in shrimp immune system and could also help in developing strategy for disease defence in shrimp.