Cloning and expression analysis of <italic>CYP</italic>302<italic>a</italic>1 from <italic>Scylla paramamosain</italic>

Abstract

In arthropods, periodic molting plays an important role in the growth, reproduction and morphogenesis. Periodic ecdysis is affected by a variety of environmental and own factors, the most important of which is controlled by the concentration of steroid hormones, and ecdysone (Ecd) whose mainly active form is 20-hydroxyecdysone(20E) is the most important steroid hormone. Moreover, ecdysteroids play important roles in regulating the growth, development and reproduction, CYP302al is the key enzyme in biosynthesis of the ecdysteriods. We obtained a full-length of <italic>CYP</italic>302<italic>a</italic>1 cDNA sequence from the mud crab <italic>Scylla paramamosain</italic> firstly, which was named as <italic>Sp</italic>-<italic>CYP</italic>302<italic>a</italic>1 in present study. The length of <italic>Sp</italic>-<italic>CYP</italic>302<italic>a</italic>1 was 3 032 bp, with a predicted 1 617 bp open reading frame (ORF) encoding 538 amino acids with a predicted molecular weight of 61.14 kDa, a 5′UTR of 205 bp and a 3′UTR of 1 210 bp. And the putative protein sequence of <italic>Sp</italic>-<italic>CYP</italic>302<italic>a</italic>1 contained 5 conserved domains including helix-C, helix-K, helix-I, PERF and heme-binding. Moreover, we found that there were no signal peptide sequences and transmembrane domains in the the putative protein sequence by Signal 5.0 Server and TMHMM. The phylogenetic tree showed that <italic>CYP</italic>302<italic>a</italic>1 of <italic>S</italic>. <italic>paramamosain</italic> was clustered with that of <italic>Portunus trituberculatus</italic> and <italic>Penaeus vannamei</italic>, and then together with other species. The amino acid sequence deduced by the <italic>Sp</italic>-<italic>CYP</italic>302<italic>a</italic>1 sequence was clustered with <italic>CY</italic>302<italic>A</italic>1 of <italic>Portunus trituberculatus</italic> and <italic>Penaeus vannamei</italic> into a small branch, and then clustered with <italic>CY</italic>302<italic>A</italic>1 of other species, and then clustered with the amino acids of <italic>CYP</italic>306<italic>A</italic>1, <italic>CYP</italic>307<italic>A</italic>1, <italic>CYP</italic>315<italic>A</italic>1, <italic>CYP</italic>314<italic>A</italic>1 and <italic>CYP</italic>18<italic>A</italic>1 encoded by other members of the <italic>P</italic>450 genes. We analyzed the expression pattern by real-time PCR (qRT-PCR). The results showed that transcript of <italic>CYP</italic>302<italic>a</italic>1 was mainly detected in the Y-organ (YO) and far exceeded other tissues both in males and females. And during the larval development, the expression of <italic>Sp</italic>-<italic>CYP</italic>302<italic>a</italic>1 kept fluctuating from zoea I to the second juvenile crab stage with the highest expression in Z3 stage. Furthermore, during molting process, the levels of <italic>CYP</italic>302<italic>a</italic>1 in Yo were extremely low during postmolt (stages A and B), and then began to increase and reached the peak at pre-molt stage (stage D), then declined. In this study, the <italic>CYP</italic>302<italic>al</italic> gene of <italic>Scylla paramamosain</italic> was cloned and identified through sequence analysis, combined with tissue distribution, larval spatiotemporal expression, and molting cycle changes. We hypothesized that <italic>CYP</italic>302<italic>al</italic> might play an vital role in the molting of <italic>S</italic>. <italic>paramamosain</italic>.