Development of a Novel shRNA Construct pSh-IRAK-4 for Silencing of IRAK-4 Gene and Delineating TLR-Mediated Pathway in Penaeus monodon In-Vitro
Ranjeeta Kumari1, Madhusudhana Rao B2, Gulshan Kumar2,
Gireesh Babu P2, Gayatri Tripathi2, KV Rajendran2
1Fisheries College & Research Institute, TNJFU, Thoothukudi- 628008
2Central Institute of Fisheries Education (Deemed University) ICAR, Panch Marg, Off Yari Road, Mumbai – 400 061
*Corresponding Author: Megha Kadam Bedekar, Aquatic Environment and Health
Management Division, ICAR-CIFE, Mumbai, India.
October 03, 2022; Published: October 17, 2022
In order to understand the TLR pathway defence mechanism of Penaeus monodon, the essential molecule involved in the transduction of Toll-pathway, Interleukin-1 (IL-1) receptor-associated kinase-4 (IRAK-4) was investigated by the mechanism of RNA interference (RNAi) by silencing of the mRNA expression of IRAK-4 gene. In this study we have developed short hairpin RNA (shRNA) construct in pSUPER vector targeting IRAK-4 gene silencing of TLR pathway in P. monodon. The silencing efficiency of pSh-IRAK-4 construct was first confirmed in-vitro in primary haemocyte culture by transfection using pSh-IRAK-4 plasmid, followed by induction with Lipopolysaccharides (LPS). Loss of expression of IRAK-4 was studied by relative gene expression in pSH-IRAK-4 group compared to LPS induced group, the maximum suppression of IRAK-4 gene in cell culture was recorded as 96% at 12h and 93.5% at 24h post LPS induction in pSh-IRAK-4 group. After confirmation of silencing efficiency of construct, the expression of TLR genes of IRAK-4 mediated pathway, was studied post LPS induction both in vitro using real-time qRT-PCR with β-actin as the internal reference gene. For this IRAK-4 downstream genes TRAF6, Dorsal and 4 Antimicrobial peptides (AMPs) (ALF, PEN, AST, Crustin) molecules were studied. Significant downregulation of mRNA expression level in downstream molecules of TLR pathway below IRAK-4 gene viz., TRAF6, Dorsal, and 4 AMPs (ALF, PEN, AST, Crustin) compared to LPS group in response to LPS stimulation was observed in pSh-IRAK-4 group (P ≤ 0.05). Taking all these results together, it is confirmed that TLR pathway is governed by central mediator kinase molecule IRAK-4, when induced by LPS ligand, NF-kB activates the downstream cascades of AMPs of the Toll pathway in P. monodon. Our result confirms the designing of a novel pSh-IRAK-4 construct and its application in efficient silencing of IRAK-4 gene in P. monodon. Plasmid-based IRAK-4 knockdown approach would provide an insight to the role of IRAK-4 in shrimp immune system.
Keywords: RNA Interference; pSUPER Vector; shRNA; IRAK-4
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