| 马氏珠母贝NatF基因克隆及其表达分析 |
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| 引用本文： | 卢金昭,房晓宸,梁海鹰,何军军,申铖皓.马氏珠母贝NatF基因克隆及其表达分析[J].南方农业学报,2021,52(11):3085-3092. |
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| 作者姓名： | 卢金昭 房晓宸 梁海鹰 何军军 申铖皓 |
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| 作者单位： | 1 广东海洋大学水产学院, 广东湛江 524088;2 广东海洋大学深圳研究院, 广东深圳 518108;3 广东省水生动物健康评估工程技术研究中心, 广东深圳 518108 |
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| 基金项目： | 国家自然科学基金项目（31472306）；广东省自然科学基金项目（2021A1515010962）；广东省海港建设与渔业产业发展专项（A201608B15）；深圳市科技计划项目（JCYJ20180507183240459） |
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| 摘 要： | 【目的】掌握马氏珠母贝（Pinctada fucata martensii）Nα-乙酰转移酶60基因（PmNatF）在不同组织、不同发育时期及不同病原相关分子模式（PAMPs）刺激下的表达变化规律，为后续研究NatF基因功能及揭示其在刺激应答中的分子机制提供理论依据。【方法】运用RACE克隆PmNatF基因cDNA序列，通过ProtScale、ProtParam、PSITE-Search、SignalP 4.1、SMART及Cell-PLoc 2.0 Package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PmNatF基因组织表达分布特征及其在不同发育时期和PAMPs刺激后的表达情况。【结果】PmNatF基因cDNA序列全长1142bp，其开放阅读框（ORF）为693 bp，5'端非编码区（5'-UTR）为181 bp，3'端非编码区（3'-UTR）为268 bp，共编码230个氨基酸残基。PmNatF蛋白分子量约26.64 kD，理论等电点（pI）为8.54，总平均亲水性系数为-0.068，为不稳定的亲水蛋白；PmNatF蛋白不存在信号肽和跨膜结构域，包含有N-乙酰转移酶结构域（Acetyltransf_1 domain），其亚细胞定位于细胞质。PmNatF蛋白二级结构中，α-螺旋占36.52%，β-转角占6.96%，延伸链占22.91%，无规则卷曲占33.91%；其三维结构与太平洋牡蛎（Crassostrea gigas）的NatF蛋白结构相似。PmNatF氨基酸序列与其他物种的NatF氨基酸序列高度同源，其中与美洲牡蛎（C.virginica）和欧洲大扇贝（Pecten maximus）的NatF氨基酸序列相似性较高，分别为76.52%和75.22%。PmNatF基因在马氏珠母贝各组织中均有表达，以在性腺中的相对表达量最高；在不同发育时期也均有PmNatF基因表达，其相对表达量以卵的最高，担轮幼虫的最低。在脂多糖（LPS）刺激下，马氏珠母贝鳃组织PmNatF基因表达呈上调趋势，于刺激24 h时达最高值；在肽聚糖（PGN）刺激下，至刺激6 h时出现明显的峰值；在聚肌胞苷酸（PolyI:C）刺激下，则在刺激72 h时出现峰值。【结论】PmNatF基因具有较高的保守性，在马氏珠母贝各组织及不同发育时期均有表达，尤其以性腺和卵的相对表达量最高，且经PAMPs刺激后在马氏珠母贝鳃组织中呈明显的差异表达，表明PmNatF基因可能参与马氏珠母贝生殖细胞的分裂和成熟及其免疫应答过程。
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| 关 键 词： | 马氏珠母贝 NatF基因 性腺 卵 PAMPs刺激 表达特征 |
| 收稿时间： | 2020-10-26 |
Cloning and expression analysis of NatF gene from Pinctada fucata martensii |
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| LU Jin-zhao,FANG Xiao-chen,LIANG Hai-ying,HE Jun-jun,SHEN Cheng-hao.Cloning and expression analysis of NatF gene from Pinctada fucata martensii[J].Journal of Southern Agriculture,2021,52(11):3085-3092. |
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| Authors: | LU Jin-zhao FANG Xiao-chen LIANG Hai-ying HE Jun-jun SHEN Cheng-hao |
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| Affiliation: | 1 Fisheries College, Guangdong Ocean University, Zhanjiang, Guangdong 524088, China;2 Shenzhen Research Institute, Guangdong Ocean University, Shenzhen, Guangdong 518108, China;3 Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment, Shenzhen, Guangdong 518108, China |
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| Abstract: | 【Objective】 By exploring the expression pattern of different tissues, different development stages, and pathogen-related molecular model(PAMPs) stimulation of Pinctada fucata martensii would provide a theoretical basis for the follow-up study of the N acetyltransferase 60(NatF) gene function and the molecular mechanism of response to stimulation.【Method】 The full length of NatF from P. fucata martensii(PmNatF) cDNA was obtained using rapid amplification of cDNA ends(RACE) technology. Bioinformatics analysis was carried out by online softwares such as ProtScale, ProtParam, PSITE-Search, SignalP 4.1, SMART, and Cell-PLoc 2.0 Package. And real-time fluorescence quantitative PCR (RT-PCR) was used to detect the expression of different tissues, different developmental stages, and performance after being stimulated by PAMPs of PmNatF.【Result】 The results showed that the full length of PmNatF cDNA was 1142 bp, containing a 5' end non coding region(5'-UTR) of 181 bp, a 3' end noncoding region(3'-UTR) of 268 bp and an open reading frame(ORF) of 693 bp which encoded 230 amino acids. It was predicted that its molecular weight of PmNatF protein was about 26.64 kD and its theoretical isoelectric point(pI) was 8.54.The overall average hydrophilicity coefficient was -0.068, which was an unstable hydrophilic protein. Analysis of deduced amino acids showed that it had no signal peptide and transmembrane domain, and contained an acetyltransf_1 domain, and subcellular localization in the cytoplasm. In the secondary structure of PmNatF protein, α-helix accounted for 36.52%, β-turn accounted for 6.96%, extended chain accounted for 22.91%, and random coil accounted for 33.91%. Its three-dimensional structure was similar to that of Pacific oyster(Crassostrea gigas) NatF protein. The amino acid sequence of PmNatF was highly conservative among species, among which the amino acid sequence similarity with the NatF of American oyster(C. virginica) and European scallop (Pecten maximus) were high, 76.52% and 75.22%, respectively. RT-PCR showed that PmNatF was expressed in all the tested tissues, with the highest expression in gonads, and the expression was also found in different developmental stages, with highest expression in the egg stage and lowest in trochophore stage. After stimulated by lipopolysaccharide(LPS), the relative expression reached the highest level at 24 h;under peptidoglycan(PGN) stimulation, PmNatF was greatly up-regulated, peaked at 6 h;under PolyI:C stimulation, the relative expression reached the highest at 72 h.【Conclusion】 The PmNatF gene is highly conserved and expresses in various tissues and in different developmental stages, especially the highly expresses in gonads and egg stage. It is differentially expressed in the gill tissue after stimulation with PAMPs, indicating that the gene may be involved in the process of in the division and maturation of germ cells and its immune response of P. fucata martensii. |
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