Isolation of Differentially Expressed Transcripts by cDNA-AFLPaba and Expression Analysis of DEAD Box ATP-Dependent RNA Helicase 48 in Ovaries of the Giant Tiger Shrimp Penaeus monodon

Abstract

Genes involved in ovarian development of the giant tiger shrimp (Penaeus monodon) were isolated by cDNA-AFLP analysis. In total, 75 primer combinations were screened against cDNA of different stages of ovaries (premature ovaries of juveniles and previtellogenic, vitellogenic and mature ovaries of wild intact and eyestalk-ablated broodstock; N = 6 for each group). Eighteen cDNA-AFLP fragments were cloned and sequenced. Nucleotide sequences of ten fragments significantly matched EF-1α (7e63), DEAD box ATP-dependent RNA helicase 48 (PmDdx48; 8e-61), tyrosine phosphatase n9 (1e-42), autophagy related protein Atg4-like protein (3e-36), nuclear pore complex protein nup154 (6e-20), GK14382 gene product (1e-20), L-3-hydroxyacryl coenzyme A dehydrogenase short chain (8e-13) and three different hypothetical proteins. The remaining fragments did not match any previously deposited sequence (E-value > 10-4). The full-length cDNA of PmDdx48 was successfully characterized. It was 1646 bp containing an open reading frame (ORF) of 1209 bp corresponding to 402 amino acids. PmDdx48 was up-regulated in late vitellogenic and mature ovaries of wild intact broodstock and in mature ovaries of wild eyestalk-ablated broodstock (P<0.05). Unilateral eyestalk ablation resulted in down-regulation of PmDdx48 in late vitellogenic and mature ovaries compared to that of wild intact broodstock (P<0.05).