拟穴青蟹CYP302a1基因的克隆及表达模式分析  

Cloning and expression analysis of CYP302a1 from Scylla paramamosain

作　　者：张林姿 赵明[1,2] 张凤英[2] 王伟[2] 刘志强 马春艳[2] 谌微[2] 王珂 马凌波[2] ZHANG Linzi;ZHAO Ming;ZHANG Fengying;WANG Wei;LIU Zhiqiang;MA Chunyan;CHEN Wei;WANG Ke;MA Lingbo(College of Fishery and Life Science, Shanghai Ocean University, Shanghai 201306, China;Key Laboratory of Oceanic and Polar Fisheries, Ministry of Agriculture and Rural Affairs , East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200090, China)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]中国水产科学研究院东海水产研究所,农业农村部远洋与极地渔业创新重点实验室,上海200090

出　　处：《海洋渔业》2021年第1期31-41,共11页Marine Fisheries

基　　金：甬财政发[2019]1045号(1019B10010);上海市科技创新行动(18391900100);现代农业产业技术体系专项(CARS-48)。

摘　　要：蜕皮激素对节肢动物生长、发育和繁殖有重要调控作用,细胞色素P450(CYP)302al是蜕皮激素合成通路中的关键酶。克隆获得了拟穴青蟹(Scylla paramamosain)CYP302a1基因,命名为Sp-CYP302a1。Sp-CYP302a1的cDNA序列长为3032 bp,包含一个1617 bp的开放阅读框(ORF),可编码538个氨基酸,预测相对分子质量为61.14 kDa。序列分析结果显示,该氨基酸含helix-K、helix-C、helix-I、PERF及heme-binding 5个P450特征保守区域。系统进化分析表明,Sp-CYP302A1与其他物种的CYP302Al聚为一类,与三疣梭子蟹(Portunus trituberculatus)和凡纳滨对虾(Penaeus vannamei)的进化关系最近。qRT-PCR分析发现,雌蟹和雄蟹中CYP302a1均在Y器官具有最高表达,而在其他组织中的表达量相对较低。在幼体发育过程中,CYP302a1在蚤状幼体Ⅲ期出现一个表达峰值,但在其他几个时期的表达量均较低。在蜕皮周期内,CYP302a1表达量变化显著,在蜕皮后期(A、B期)表达量最低,随后开始上升,至蜕皮前期(D期)达到最大值,随后开始下降。综上,研究从序列分析方面鉴定了拟穴青蟹的CYP302a1,并对该基因进行了表达模式综合分析,结果表明其可能在拟穴青蟹蜕皮以及幼体发育调控中具有重要作用。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 3032 bp,with a predicted 1617 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 1210 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.Furt

关 键 词：拟穴青蟹 CYP302a1 表达模式分析 蜕皮周期 蜕皮激素 

分 类 号：S917.4[农业科学—水产科学]