| 马氏珠母贝基质金属蛋白酶基因MMP-17的克隆及表达分析 |
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| 引用本文： | 罗少杰,闫芳,郑哲,田荣荣,邓岳文,焦钰.马氏珠母贝基质金属蛋白酶基因MMP-17的克隆及表达分析[J].水产学报,2015,39(7):978-988. |
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| 作者姓名： | 罗少杰 闫芳 郑哲 田荣荣 邓岳文 焦钰 |
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| 作者单位： | 广东海洋大学水产学院, 广东 湛江 524025,广东海洋大学水产学院, 广东 湛江 524025,广东海洋大学水产学院, 广东 湛江 524025,广东海洋大学水产学院, 广东 湛江 524025,广东海洋大学水产学院, 广东 湛江 524025;广东省珍珠养殖与加工工程技术研究中心, 广东 湛江 524025,广东海洋大学水产学院, 广东 湛江 524025 |
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| 基金项目： | 国家自然科学基金(31272635,31372526,41206141);广东海洋大学博士启动项目(1212318) |
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| 摘 要： | 基质金属蛋白酶(matrix metalloproteinases,MMP)是一种能够降解细胞外基质的蛋白水解酶。MMP-17是一种膜型基质金属蛋白酶,通过糖基磷脂酰肌醇连接于细胞表面,参与调控有机体的内环境稳定、宿主防御等多种生理过程。为研究MMP-17在马氏珠母贝免疫反应中的作用,实验运用RACE技术,克隆得到马氏珠母贝MMP-17(Pinctada martensii MMP,Pm-MMP-17)基因cDNA 全长序列,并对其序列特征及功能进行初步分析。结果显示,Pm-MMP-17基因cDNA全长2 794 bp,开放阅读框(ORF)为1 923 bp,编码640个氨基酸,5'UTR长156 bp,3'UTR长715 bp,分子量约为73.11 ku,等电点为8.98;多序列比对和系统进化树分析表明,Pm-MMP-17与其他物种的MMP具有较高的保守性,与长牡蛎的MMP-17相似性高达82%;功能结构域分析表明,Pm-MMP-17有5个高度保守的结构区域:N-末端的信号肽、前导区、催化区、铰链区和C-末端的类血红素结合蛋白区;荧光定量数据分析表明,Pm-MMP-17基因在马氏珠母贝的闭壳肌、珍珠囊、足、外套膜、血淋巴、肝胰腺、性腺、鳃等8个组织中均有表达,在血液中的表达量最高,闭壳肌和鳃次之;脂多糖(LPS)刺激后,Pm-MMP-17基因表达水平上调,12 h后达到最大值,之后又逐渐下调并恢复到正常水平。研究表明,Pm-MMP-17基因可能在马氏珠母贝的免疫反应中起着重要作用。
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| 关 键 词： | 马氏珠母贝 Pm-MMP-17基因 克隆 荧光定量 表达分析 |
| 收稿时间： | 2015/1/24 0:00:00 |
| 修稿时间： | 2015/5/28 0:00:00 |
Molecular cloning and expression analysis of matrix metalloproteinase 17 gene from Pinctada martensii |
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| LUO Shaojie,YAN Fang,ZHENG Zhe,TIAN Rongrong,DENG Yuewen and JIAO Yu.Molecular cloning and expression analysis of matrix metalloproteinase 17 gene from Pinctada martensii[J].Journal of Fisheries of China,2015,39(7):978-988. |
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| Authors: | LUO Shaojie YAN Fang ZHENG Zhe TIAN Rongrong DENG Yuewen and JIAO Yu |
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| Institution: | Fisheries College, Guangdong Ocean University, Zhanjiang 524025.China,Fisheries College, Guangdong Ocean University, Zhanjiang 524025.China,Fisheries College, Guangdong Ocean University, Zhanjiang 524025.China,Fisheries College, Guangdong Ocean University, Zhanjiang 524025.China,Fisheries College, Guangdong Ocean University, Zhanjiang 524025.China;Guangdong Technology Research Center for Pearl Aquaculture and Process, Zhanjiang 524025, China and Fisheries College, Guangdong Ocean University, Zhanjiang 524025.China |
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| Abstract: | Matrix metalloproteinases(MMPs)could degrade the extracellular matrix and participate in various physiological processes,such as homeostasis and immune response.MMP-17 is one membrane type MMP that attaches to the cell surface by Glycosyl-phosphatidyl inositol(GPI),and participates in various physiological processes,such as organic homeostasis and immune defense.The pearl oyster Pinctada martensii is the main species cultured for marine pearl production in China.To deliberate the function of MMP-17 in the immune response of P.martensii,in this study,using rapid amplification of cDNA ends(RACE)technology,we have obtained the full length sequence of Pinctada martensii MMP(Pm-MMP-17)cDNA and analyzed its sequence characteristics and function.Our results showed that the cDNA full length of Pm-MMP-17 is 2 794 bp,including 156 bp of 5'UTR,715 bp of 3'UTR,and 1 923 bp of open reading frame(ORF)encoding 640 amino acid residues with an estimated molecular mass of 73.11 ku and theoretical isoelectric point of 8.98.Multi-sequence alignment and phylogenetic analysis results showed that Pm-MMP-17 was highly homologous with that from other species and had 82% sequence identity with Crassostrea gigas MMP-17.Amino acid sequence analysis showed Pm-MMP-17 had five conservative domains:signal peptide,propeptide,catalytic domain,hinge region and hemopexin-like domain.Meanwhile,real-time PCR analysis showed that Pm-MMP-17 was ubiquitously expressed in all tissues detected,including adductor muscle,pearl sac,foot,mantle,hemolymph,hepatopancreas,gonads and gills,with the highest expression level in hemolymph,followed by adductor muscle and gill.Previous research has showed that Pm-MMP-17 was highly expressed in the eosinophils and involved in the regulation of cell migration,growth and differentiation.We supposed that the highly expression of Pm-MMP-17 gene in the hemolymph was crucial for the migration of hemolymph in the body of pearl oyster.After LPS stimulation,the expression level of Pm-MMP-17 began to increase and reached the highest level at 12 h,and then gradually declined to the normal level(P<0.05),indicating again the important function of Pm-MMP-17 gene in the immune response of pearl oyser P.martensii.Therefore,our data showed Pm-MMP-17 participated in the immune response in P.martensii and provided the basis for further research in the mechanism underlying the immune defense in pearl oyster. |
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| Keywords: | Pinctada martensii Pm-MMP-17 gene cloning real-time PCR expression analysis |
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