| 罗氏沼虾细胞色素P450家族CYP302a1基因克隆及其在蜕皮周期中的表达 |
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| 引用本文： | 杨光,秦真东,赵丽娟,詹凡玢,沈海洋,张梦兰,卢志杰,叶成凯,李凤麟,潘淦,林蠡.罗氏沼虾细胞色素P450家族CYP302a1基因克隆及其在蜕皮周期中的表达[J].水产学报,2020,44(4):562-574. |
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| 作者姓名： | 杨光 秦真东 赵丽娟 詹凡玢 沈海洋 张梦兰 卢志杰 叶成凯 李凤麟 潘淦 林蠡 |
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| 作者单位： | 华南师范大学生命科学学院,广东省水产健康安全养殖重点实验室,广东广州 510631;仲恺农业工程学院动物科技学院,广东省水环境与水产品安全工程技术研究中心,广州市水产病害与水禽养殖重点实验室,广东广州 510225;仲恺农业工程学院动物科技学院,广东省水环境与水产品安全工程技术研究中心,广州市水产病害与水禽养殖重点实验室,广东广州 510225;华南师范大学生命科学学院,广东省水产健康安全养殖重点实验室,广东广州 510631 |
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| 基金项目： | 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) |
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| 摘 要： | 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1859 bp,开放阅读框(ORF)为1629 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、helixC、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,MrCYP302a1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。
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| 关 键 词： | 罗氏沼虾 蜕皮周期 基因克隆 CYP302a1 组织表达 |
| 收稿时间： | 2019/7/7 0:00:00 |
| 修稿时间： | 2019/9/25 0:00:00 |
Cloning and characterization of cytochrome P450 302a1 (CYP302a1) during molting stages in Macrobrachium rosenbergii |
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| YANG Guang,QIN Zhendong,ZHAO Lijuan,ZHAN Fanbin,SHEN Haiyang,ZHANG Menglan,LU Zhijie,YE Chengkai,LI Fenglin,PAN Gan and LIN Li.Cloning and characterization of cytochrome P450 302a1 (CYP302a1) during molting stages in Macrobrachium rosenbergii[J].Journal of Fisheries of China,2020,44(4):562-574. |
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| Authors: | YANG Guang QIN Zhendong ZHAO Lijuan ZHAN Fanbin SHEN Haiyang ZHANG Menglan LU Zhijie YE Chengkai LI Fenglin PAN Gan and LIN Li |
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| Institution: | Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, Guangzhou 510631, China;Guangdong Provincial Water Environment and Aquatic Products Security Engineering Technology Research Center, Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China,Guangdong Provincial Water Environment and Aquatic Products Security Engineering Technology Research Center, Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China,Guangdong Provincial Water Environment and Aquatic Products Security Engineering Technology Research Center, Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China,Guangdong Provincial Water Environment and Aquatic Products Security Engineering Technology Research Center, Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China,Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, Guangzhou 510631, China;Guangdong Provincial Water Environment and Aquatic Products Security Engineering Technology Research Center, Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China,Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, Guangzhou 510631, China;Guangdong Provincial Water Environment and Aquatic Products Security Engineering Technology Research Center, Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China,Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, Guangzhou 510631, China;Guangdong Provincial Water Environment and Aquatic Products Security Engineering Technology Research Center, Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China,Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, Guangzhou 510631, China;Guangdong Provincial Water Environment and Aquatic Products Security Engineering Technology Research Center, Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China,Guangdong Provincial Water Environment and Aquatic Products Security Engineering Technology Research Center, Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China,Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, Guangzhou 510631, China and Guangdong Provincial Water Environment and Aquatic Products Security Engineering Technology Research Center, Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China |
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| Abstract: | 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. |
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| Keywords: | Macrobrachium rosenbergii molting stage gene cloning CYP302a1 expression |
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