| 大珠母贝组织蛋白酶B基因克隆及其表达分析 |
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| 引用本文： | 徐扬,王姿曼,李俊辉,梁飞龙,邓岳文,杨创业.大珠母贝组织蛋白酶B基因克隆及其表达分析[J].南方农业学报,2021,52(5):1353-1361. |
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| 作者姓名： | 徐扬 王姿曼 李俊辉 梁飞龙 邓岳文 杨创业 |
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| 作者单位： | 广东海洋大学水产学院,广东湛江 524088;广东省珍珠养殖与加工工程技术研究中心,广东湛江 524088;广东省珍珠科技创新中心,广东湛江 524088;广东海洋大学水产学院,广东湛江 524088;广东省珍珠养殖与加工工程技术研究中心,广东湛江 524088;广东省珍珠科技创新中心,广东湛江 524088;广东省海水养殖生物育种工程实验室,广东湛江 524088 |
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| 基金项目： | 国家贝类产业技术体系建设专项（CARS-49）；广东省教育厅创新团队建设项目（2017KCXTD016）；广东省贝类产业技术体系建设专项（2020KJ146） |
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| 摘 要： | 【目的】分析组织蛋白酶B基因（CatB）在大珠母贝（Pinctada maxima）不同组织中的表达模式，明确CatB在大珠母贝中的功能作用，为培育生长快且抗逆性强的大珠母贝提供基础资料。【方法】利用RACE克隆大珠母贝CatB基因，利用ExPASy ProtParam、ExPASy ProtScale、NPS@SOPMA、SWISS-MODEL及SignalP 4.1等在线软件进行生物信息学分析，并以实时荧光定量PCR检测CatB基因在大珠母贝外套膜（套膜区、边缘膜区和中央膜区）、肝胰腺、鳃、足和闭壳肌等组织中的表达情况。【结果】大珠母贝CatB基因cDNA序列全长1365 bp，其开放阅读框（ORF）1026 bp，5'非编码区（5'-UTR）长度81 bp，3'非编码区（3'-UTR）长度258 bp，共编码341个氨基酸残基。大珠母贝CatB蛋白分子量为37.73 kD，理论等电点（pI）为6.66，脂溶性系数为67.48，不稳定指数为31.18，亲水性平均系数（GRAVY）为-0.451，为稳定的亲水性蛋白；在第89~337位氨基酸存在一个Pept-C1结构域。大珠母贝CatB蛋白二级结构以无规则卷曲为主，占51.03%，α-螺旋占26.98%，β-转角占9.09%，延伸链占12.90%；三级结构与马氏珠母贝（P.fucata martensii）CatB蛋白结构相似。大珠母贝CatB氨基酸序列与马氏珠母贝CatB氨基酸序列（ADX32985.1）的相似性高达90.91%；与长牡蛎（Crassostrea gigas，XP_011428258.1）、海湾扇贝（Argopecten irradians，ANG56311.1）、褶纹冠蚌（Cristaria plicata，AEF32260.1）的CatB氨基酸序列相似性分别为79.47%、65.38%和62.18%。CatB基因在大珠母贝外套膜的套膜区、边缘膜区和中央膜区及肝胰腺、鳃、足和闭壳肌等7个组织中均有表达，以肝胰腺中的相对表达量最高，显著高于在其他组织中的相对表达量（P<0.05），其次是外套膜的套膜区和中央膜区。【结论】CatB基因在大珠母贝肝胰腺中高表达，其次是外套膜的套膜区和中央膜区，故推测CatB是通过参与大珠母贝的消化吸收作用而调控其生长代谢过程。
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| 关 键 词： | 大珠母贝 组织蛋白酶B(CatB) 基因克隆 组织表达 肝胰腺 消化吸收 |
| 收稿时间： | 2020-07-16 |
Cloning and expression analysis of cathepsin B gene from Pinctada maxima |
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| XU Yang,WANG Zi-man,LI Jun-hui,LIANG Fei-long,DENG Yue-wen,YANG Chuang-ye.Cloning and expression analysis of cathepsin B gene from Pinctada maxima[J].Journal of Southern Agriculture,2021,52(5):1353-1361. |
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| Authors: | XU Yang WANG Zi-man LI Jun-hui LIANG Fei-long DENG Yue-wen YANG Chuang-ye |
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| Affiliation: | 1. Fisheries College, Guangdong Ocean University, Zhanjiang, Guangdong 524088, China;2. Pearl Breeding and Processing Engineering Technology Research Center of Guangdong, Zhanjiang, Guangdong 524088, China;3. Guangdong Science and Innovation Center for Pearl Culture, Zhanjiang, Guangdong 524088, China;4. Guangdong Provincial Engineering Laboratory for Mariculture Organism Breeding, Zhanjiang, Guangdong 524088, China |
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| Abstract: | 【Objective】Analyzed the expression patterns of cathepsin B gene(CatB) in different tissues of Pinctada maxima, and clarified the functional role of CatB in P. maxima, in order to provide basic information for cultivating fastgrowing and stress-resistant P. maxima.【Method】The CatB gene in P. maxima(designated as PmCatB) was obtained by RACE with ExPASy ProtParam, ExPASy ProtScale, NPS@SOPMA, SWISS-MODEL and SignalP 4.1 online softwares for bioinformatics analysis and quantitative real-time PCR was used to detect the expression level of PmCatB in different tissues including the mantle (marginal zone of mantle, pallial zone of mantle and central zone of mantle), hepatopancreas, gill, foot and adductor muscle.【Result】The full length of PmCatB cDNA sequence was 1365 bp, with an open reading frame(ORF) of 1026 bp, 5' untranslated region(UTR) was 81 bp, 3' UTR was 258 bp, and encoded 341 amino acids residues. The molecular weight of PmCatB protein was 37.73 kD and the theoretical isoelectric point(pI) was 6.6. The fat solubility coefficient was 67.48, the instability index was 31.18, and the average hydrophilicity coefficient(GRAVY) was -0.451, which was a stable hydrophilic protein;there was a Pept-C1 domain at amino acids 89-337. The secondary structure prediction of PmCatB protein showed that random coils were dominant, accounting for 51.03%, α-helix accounted for 26.98%, β-turn accounted for 9.09%, and extended strand accounted for 12.90%; the tertiary structure was similar to the CatB protein structure of P. fucata martensii. The similarity between the CatB amino acid sequence of P. maxima and the P. f. martensii (ADX32985.1) was as high as 90.91%; and the similarities of the amino acid sequence of the Crassostrea gigas(XP_011428258.1), Argopecten irradians(ANG56311.1) and Cristaria plicata(AEF32260.1) compared with P. maximawere 79.47%, 65.38% and 62.18% respectively. PmCatB was expressed in tissues including the marginal zone of mantle, pallial zone of mantle, central zone of mantle, hepatopancreas, gill, foot and adductor muscle, the relative expression in hepatopancreas was the highest, and the relative expression level wassignificantly higher than the relative expression in other tissues(P<0.05), and followed with the marginal zone of the mantle and the central zone of the mantle.【Conclusion】PmCatB shows high expression in the hepatopancreas, and followed by the pallial zone of mantle and central zone of mantle, so it is speculated that PmCatB may participate in the process of growth and metabolism process bytaking part in the digestion and absorption of P.maxima. |
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