| 卵形鲳JunB基因克隆及其胚胎组织表达分析 |
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| 引用本文： | 潘传燕,余艳玲,罗洪林,冯鹏霏,宋漫玲,肖蕊,张永德.卵形鲳JunB基因克隆及其胚胎组织表达分析[J].南方农业学报,2021,52(11):3102-3110. |
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| 作者姓名： | 潘传燕 余艳玲 罗洪林 冯鹏霏 宋漫玲 肖蕊 张永德 |
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| 作者单位： | 广西水产科学研究院/广西水产遗传育种与健康养殖重点实验室, 南宁 530021 |
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| 基金项目： | 广西创新驱动发展专项（桂科AA17204080-3，桂科AA18242031-2）；广西水产遗传育种与健康水产养殖重点实验室自主研发项目（19-A-01-05） |
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| 摘 要： | 【目的】明确卵形鲳鲹（Trachinotus ovatus）JunB基因（ToJunB）在胚胎发育过程中的表达规律，为揭示JunB基因在鱼类胚胎发育过程中的作用机制打下基础。【方法】采用RT-PCR扩增ToJunB基因编码区（CDS）序列，通过ProtParam、NetSurfP 2.0、TMHMM、SignalP、PSORT及NetNGlyc 1.0等在线软件进行生物信息学分析，并采用荧光定量PCR检测ToJunB基因在卵形鲳鲹17个胚胎发育时期（受精卵、2-细胞期、8-细胞期、16-细胞期、32-细胞期、64-细胞期、多细胞期、高囊胚期、原肠早期、原肠中期、原肠末期、胚胎形成期、眼囊期、耳囊期、心脏跳动期、晶体出现期和初孵仔期）的表达情况。【结果】克隆获得的ToJunB基因（1777 bp）包含344 bp的5'端非编码区（5'-UTR）、954 bp的开放阅读框（ORF）及479 bp的3'端非编码区（3'-UTR），共编码318个氨基酸残基；其编码蛋白分子量为35.03 kD，理论等电点（pI）为8.26，呈碱性；总平均疏水指数（GRAVY）为-0.567，为亲水性蛋白。ToJunB蛋白无跨膜结构和信号肽，主要定位于细胞核，属于非分泌型蛋白，在第39、129和168位氨基酸处各存在1个潜在的糖基化位点；其二级结构中α-螺旋占35.67%、延伸链占14.33%、无规则卷曲占50.00%。基于JunB氨基酸序列相似性构建的系统发育进化树显示，卵形鲳鲹与黄尾鰤的遗传距离最近，均隶属于鲈形目鲹科。ToJunB基因在胚胎发育前期（受精卵至原肠早期）的相对表达量较高，至原肠中期达最高值，在胚胎发育后期（原肠末期至初孵仔期）的相对表达量均较低，且ToJunB基因在受精卵至原肠中期共10个发育时期的相对表达量极显著高于胚胎发育后期的7个时期（P<0.01）。【结论】ToJunB基因在卵形鲳鲹胚胎受精卵至原肠早期的相对表达量较高，于原肠中期达最高值后迅速降低，原肠末期至初孵仔期的相对表达量均较低，说明JunB基因在卵形鲳鲹胚胎发育的细胞分裂和增殖过程中发挥重要作用。
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| 关 键 词： | 卵形鲳鯵 JunB基因 胚胎发育 生物信息学分析 表达规律 |
| 收稿时间： | 2020-10-26 |
Cloning of JunB gene in Trachinotus ovatus and its expression in embryonic tissues |
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| PAN Chuan-yan,YU Yan-ling,LUO Hong-lin,FENG Peng-fei,SONG Man-ling,XIAO Rui,ZHANG Yong-de.Cloning of JunB gene in Trachinotus ovatus and its expression in embryonic tissues[J].Journal of Southern Agriculture,2021,52(11):3102-3110. |
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| Authors: | PAN Chuan-yan YU Yan-ling LUO Hong-lin FENG Peng-fei SONG Man-ling XIAO Rui ZHANG Yong-de |
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| Affiliation: | Guangxi Academy of Fishery Sciences/Guangxi Key Laboratory of Aquatic Genetic Breeding and Healthy Aquaculture, Nanning 530021, China |
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| Abstract: | 【Objective】 To explore the expression pattern of Trachinotus ovatus JunB gene(ToJunB) in embryonic development, and lay a foundation for exploring the mechanism of JunB on embryonic development process.【Method】 RT-PCR was used to amplify the encoding region(CDS) sequence of the ToJunB gene, and the bioinformatics analysis was carried out with online softwares such as ProtParam, NetSurfP 2.0, TMHMM, SignalP, PSORT and NetNGlyc 1.0. Real-time fluorescence quantitative PCR was used to detect the expression of ToJunB mRNA in 17 bryonic development stages of T. ovatus(zygote, 2-cell embryo, 8-cell embryo, 16-cell embryo, 32-cell embryo, 64-cell embryo, multicellular stage, blastula stage, early gastrula stage, gastrula stage, late gastrula stage, embryo formed stage, optic vesicle stage, otocyst vesicle stage, heart pulsation stage, formation of eye lens and hatching stage).【Result】 The ToJunB gene cloned from embryonic tissues of T. ovatus was 1777 bp in length, consisted of 344 bp 5' end non-coding region(5'- UTR), 954 bp open reading frame(ORF) and 479 bp 3' end non-coding region(3'-UTR), encoding 318 amino acid residues, its protein molecular weight was 35.03 kD, theoretical isoelectric point (pI) was 8.26, it was alkaline. The total average hydrophobic index(GRAVY) was -0.567, indicating that it belonged to hydrophilic protein. ToJunB protein had no transmembrane structure, no signal peptide sequence, and a potential glycosylation site at amino acid 39, 129, 168. The ToJunB protein mainly existed in the nucleus, belonging to asecretory protein. In the secondary structure, α- helix and extended strand accounted for 35.67% and 14.33%, random coil accounted for 50.00%. The phylogenetic trees based on amino acid sequence similarity of JunB indicated that the closest genetic distance with T. ovatus and Serio lalalandi dorsalis. They both were Hericidae, Perciformes. The relative expression of ToJunB gene was higher in the early stage of embryonic development(from the zygote to the early gastrula stage), reached the highest value in the gastrula stage, and kept low value in the late stage of embryonic development(from the late gastrula stage to the hatching stage). The expression level of ToJunB gene in the 10 developmental stages from zygote to early gastrula was extremely significantly higher than that from the late gastrula to the early hatching stage(P<0.01).【Conclusion】 The relative expression of ToJunB gene is higher from the zygote to the early gastrula stage, reaches the highest value in the gastrula stage and then reduces rapidly, and keeps low value from the late gastrula stage to the hatching stage. The result show that JunB gene plays an important role in the cell division and proliferation in embryo development of T. ovatus. |
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