蜕皮是甲壳动物重要的生理活动，与其蜕皮激素的合成密切相关，细胞色素P450(CYP)302a1是甲壳动物蜕皮激素合成通路中的关键酶之一。本研究克隆了罗氏沼虾CYP302a1基因(Mr-CYP302a1)，cDNA全长1 859 bp，开放阅读框(ORF)为1 629 bp，编码543个氨基酸(aa)，分子量大小为61.09 ku，等电点为8.42。氨基酸序列分析显示CYP302a1基因的保守结构域含有5个P450基因家族特征保守区域：heme-binding、helix-K、helix-C、helix-I及PERF。系统进化分析结果显示Mr-CYP302a1首先与绿虾CYP302a1聚为一支，然后与凡纳滨对虾及三疣梭子蟹等十足目甲壳动物的CYP302a1聚为一支，与甲壳动物的亲缘关系最近。实时荧光定量PCR(qRT-PCR)检测表明Mr-CYP302a1在罗氏沼虾的多个组织中均有表达，其中在Y器官中的表达量最高，性腺中次之。同时研究发现，Mr-CYP302a1基因在罗氏沼虾的蜕皮后期(A期和B期)表达量很低，蜕皮间期(C期)表达量开始上升，在蜕皮前期D₁亚期达到峰值。对Mr-CYP302a1进行蛋白表达及多克隆抗体制备，蛋白印迹法(Western blot，WB)检测表明Mr-CYP302a1蛋白在罗氏沼虾Y器官中的表达量最高，在蜕皮过程中的蜕皮前期D₁亚期达到峰值。综上所述，该基因在罗氏沼虾的蜕皮过程中扮演着十分重要的角色。

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.

Q785;S917.4

国家自然科学基金(31872606)；广东省海洋与渔业局基金(GDME-2018C006，D21822202)；中国—东盟海上合作基金(CAMC-2018F)；广东省教育厅基金(KA170500G，TK222001G，KA18058B3，KA1819604)；广东省现代农业产业技术体系创新团队建设专项(2019KJ141)