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

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 CYP302a1 cDNA sequence from the mud crab Scylla paramamosain firstly, which was named as Sp-CYP302a1 in present study. The length of Sp-CYP302a1 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 Sp-CYP302a1 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 CYP302a1 of S. paramamosain was clustered with that of Portunus trituberculatus and Penaeus vannamei, and then together with other species. The amino acid sequence deduced by the Sp-CYP302a1 sequence was clustered with CY302A1 of Portunus trituberculatus and Penaeus vannamei into a small branch, and then clustered with CY302A1 of other species, and then clustered with the amino acids of CYP306A1, CYP307A1, CYP315A1, CYP314A1 and CYP18A1 encoded by other members of the P450 genes. We analyzed the expression pattern by real-time PCR (qRT-PCR). The results showed that transcript of CYP302a1 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 Sp-CYP302a1 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 CYP302a1 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 CYP302al gene of Scylla paramamosain was cloned and identified through sequence analysis, combined with tissue distribution, larval spatiotemporal expression, and molting cycle changes. We hypothesized that CYP302al might play an vital role in the molting of S. paramamosain.