Molecular characterisation, ontogeny and tissue level expression analysis of a downstream signalling molecule of the Toll-pathway, Dorsal (an NF-κB homologue) in black tiger shrimp (Penaeus monodon).

Abstract

Transformer 2 (tra 2) and fruitless (fru) genes have been proven to play a key role in sex determination pathways in many Arthropods, including insects and crustaceans. In this study, a paralog of P. monodon tra 2 (Pmtra 2), P. monodon ovarian associated transformer 2 (PmOvtra 2) and 2 isoforms of P. monodon fruitless-like gene (Pmfru-1 and Pmfru-2) were identified and characterized. The full cDNA sequence of PmOvtra 2 consisted of 1,774 bp with the longest open reading frame (ORF) of 744 bp encoding for 247 amino acids. The PmOvtra 2 exhibited a predicted RNA-recognition motif (RRM) domain and two arginine-serine (RS) regions, suggesting its function in RNA splicing. The full cDNA sequence of Pmfru-1 consisted of 1,306 bp with 1,182 bp ORF encoding for 393 amino acids, whereas the full cDNA sequence of Pmfru-2 consisted of 1,858 bp with 1,437 bp ORF encoding 478 amino acids. The deduced amino acid sequences of Pmfru-1 and Pmfru-2 exhibited highly conserved domains of Fru proteins, including Broad-complex, Tramtrack and Bric-a-brac (BTB), and zinc finger (ZF) domains. In addition, Pmfru-1 and Pmfru-2 were suggestively originated from the same single genomic locus by genomic sequence analysis. Specifically, Pmfru pre-mRNA was alternatively spliced for Pmfru-1 and Pmfru-2 to include mutually exclusive exon 7 and exon 6, respectively. Temporal and spatial expression of PmOvtra 2, Pmfru-1, and Pmfru-2 were also investigated by qPCR. The results showed that all were expressed in early developmental stages with undifferentiated gonads starting from nauplius until postlarvae. The expression of PmOvtra 2 started at nauplius stage and gradually increased from mysis to postlarvae (PL) 1. However, the expression of Pmfru-1 was low at the nauplii stage and slightly increased from protozoea to PL5, whereas the expression of Pmfru-2 maintained a low level from nauplius to mysis and then gradually increased at the PL stages. Expressions of PmOvtra 2, Pmfru-1, and Pmfru-2 were detected in various tissues including nervous tissue, gill, heart, hepatopancreas, gut, and gonads. Interestingly, the sexually dimorphic expression of PmOvtra 2, Pmfru-1, and Pmfru-2 was demonstrated in fully developed gonads in which the ovary showed significantly higher expressions than the testis. The great difference in the expression pattern of PmOvtra 2, Pmfru-1, and Pmfru-2 in the ovary and testis suggested their roles in the female sex determination in P. monodon.