Abstract:Ecdysteroids in crustaceans are mainly synthesized and secreted in the Y-organs（YO），and involve in the many essential physiological activities such as molting，reproduction，etc. The cytochrome P450（CYP）307a1 encoded by Spook is the key enzyme in early steps of ecdysteroids biosynthesis. To study the regulatory role of Spook in molting process，the full-length cDNA of gene was cloned and characterized from Portunus trituberculatus（GenBank accession number：KM030021）by reverse transcription PCR（RT-PCR）and RACE，designated as Pt-Spook. The cDNA of Pt-Spook was 2 200 bp in length and contained an opening reading frame（ORF）of 1 563 bp encoding 520 amino acid residues. Multiple alignment revealed that the Pt-Spook contained 5 conserved domains of helix-C，helix-K，helix-I，PERF and heme-binding. The analysis by phylogenetic tree discovered that the Pt-Spook clustered in the same branch with other species，however，Halloween genes were in another branch，indicating that the deduced amino acid sequence was truly the protein sequence of P. trituberculatus Spook. The tissue distribution of Pt-Spook was detected by qPCR，and the results showed that Pt-spook expression was highly specific to YO. During the molt cycle，the expression of Pt-spook gradually increased from post-molt（stage A and B），and reached the maximum in intermolt（stage C），then gradually decreased during pre-molt to the minimum in sub-stage D3 and D4. The results suggested that the Pt-spook involved in the regulation of molt cycle in P. trituberculatus.