Cloning and characterization of cytochrome P450 302a1 (CYP302a1) during molting stages in Macrobrachium rosenbergii

The molting process is an essential physiological process in crustaceans that is closely related to the synthesis of ecdysteriods. Cytochrome P450(CYP)302a1 is the key enzyme which plays a critical role in the synthesis of ecdysteriods. Here we present the cloning and characterization of CYP302a1 gene from Macrobrachium rosenbergii (Mr-CYP302a1). The acquired gene was 1 859 bp in full-length with the open reading frame (ORF) of 1 629 bp that encodes 543 amino acids (aa) with a molecular weight of 61.09 ku and an isoelectric point of 8.42. The aa sequence analysis revealed that there were five P450 characteristic conserved regions, i.e., heme-binding, helix-K, helix-C, helix-I, and PERF. Phylogenetic analysis demonstrated that Mr-CYP302a1 was closely related to the CYP302a1 of Neocaridina denticulata, and then clustered with the CYP302a1 from Decapoda crustaceans such as Litopenaeus vannamei and Portunus trituberculatus. Real-time quantitative PCR (qRT-PCR) results showed that Mr-CYP302a1 was expressed in almost all the tissues tested with significantly higher expression levels in the Y-organ. On the other hand, the expression of Mr-CYP302a1 was significantly lower at the postmolt stage (stages A and B), and it was increased gradually at the intermolt (stage C), significantly enhanced and reached the maximal level at the D₁ stage. Mr-CYP302a1 was expressed and its polyclonal antibody was generated. Western blot (WB) showed that the expression of Mr-CYP302a1 protein was the highest in Y- organs of M. rosenbergii. The expression level of Mr-CYP302a1 protein also reached a peak at D₁ stage during the molting process. In summary, our results indicate that Mr-CYP302a1 may play an important role in molting of M. rosenbergii.