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细胞色素P450(CYP)302a1是昆虫蜕皮激素合成通路中的关键酶,为了研究其在三疣梭子蟹蜕皮过程中的调控作用,实验采用反转录PCR(RT-PCR)和c DNA末端快速扩增(RACE)技术,克隆得到了三疣梭子蟹CYP302a1全长c DNA序列(Gen Bank登录号:KM596851)。该序列全长为3 171 bp,包含一个长度为1 626 bp的开放阅读框,编码541个氨基酸;序列分析显示该氨基酸含helix-C、helix-K、helix-I、PERF及heme-binding共5个P450特征保守区域;系统进化树分析发现三疣梭子蟹CYP302a1与日本剑水蚤CYP302a1聚为一小支,再与其他物种CYP302a1聚为一支。采用实时荧光定量PCR(qRT-PCR)技术分析了CYP302a1在不同组织和蜕皮过程中的表达水平变化。结果显示,CYP302a1的表达量在Y器中最高,而在其他组织中均极低(P<0.05);蜕皮过程中,CYP302a1在蜕皮后期(A、B期)表达量最低,从蜕皮间期(C期)开始上升,至蜕皮前期的D1亚期达到最大值,随后下降。结果表明CYP302a1在三疣梭子蟹蜕皮调控中可能起着重要作用。
Cytochrome P450 (CYP) 302a1 is a key enzyme in the ecdysone synthesis pathway. In order to study its regulation in molting of Portunus trituberculatus, reverse transcription PCR (RT-PCR) and rapid amplification of c DNA end (RACE) technique, the full length c DNA sequence of CYP302a1 was cloned from Portunus tritubus (Gen Bank Accession No. KM596851). The full length of this sequence was 3 171 bp and contained an open reading frame with a length of 1 626 bp encoding 541 amino acids. Sequence analysis showed that the amino acids contained helix-C, helix-K, helix-I, PERF and heme-binding Five P450 conserved regions were detected. Phylogenetic tree analysis showed that CYP302a1 of Portunus trituberculatus and CYP302a1 of S. japonicus clustered into a small branch, and then clustered together with other species CYP302a1. The expression level of CYP302a1 in different tissues and molting process was analyzed by real-time fluorescence quantitative PCR (qRT-PCR). The results showed that the expression level of CYP302a1 was the highest in Y and the lowest in other tissues (P <0.05). During the ecdysis, the expression level of CYP302a1 was the lowest during the late eclampsia stage (A, B) C phase) began to rise, to pre-molting D1 subphase reached its maximum, then decreased. The results showed that CYP302a1 may play an important role in the regulation of molting of Portunus trituberculatus.