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| 秋茄KoNAC25基因的克隆、亚细胞定位及表达分析 |
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杨党1,2, 蒋文骏1, 王永峰3, 王建强4, 董超4, 杜照奎1,2*
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(1.台州学院 生命科学学院,浙江 台州 318000；2. 台州学院 浙江省植物进化生态学与保护重点实验室, 浙江 台州 3180001；3. 台州循环经济发展有限公司, 浙江 台州 318000； 浙江省水文地质工程地质大队, 浙江 宁波 315000
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| 摘要: |
| NAC是植物所特有的转录因子,在调节植物生长发育、激素信号转导和逆境胁迫响应等方面发挥着重要作用。前期工作中,从红树植物秋茄(Kandelia obovata)基因组中鉴定到一个受低温诱导的NAC基因KoNAC25 (GenBank登录号PP860407)。为探究秋茄KoNAC25基因是否参与盐胁迫条件下的响应,该研究通过 RT-PCR技术从秋茄叶片cDNA中克隆到KoNAC25基因的开放阅读框(ORF),并对其进行生物信息学、亚细胞定位及基因表达分析等。结果表明：(1)秋茄KoNAC25基因的编码区全长为858 bp,共编码285个氨基酸,分子量为32.9 kDa,理论等电点为8.53,为亲水性蛋白,不含信号肽且无跨膜结构。(2) KoNAC25蛋白N端具有一个NAM超家族结构域,由五个保守的亚结构域构成,属于NAM亚家族成员；系统进化树表明秋茄KoNAC25转录因子与大戟科的蓖麻(Ricinus communis)、木薯(Manihot esculenta)和橡胶树(Hevea brasiliensis)等植物亲缘关系较近。(3)亚细胞定位试验证实该蛋白定位于细胞核,qRT-PCR分析结果表明,KoNAC25基因在叶中表达量显著高于花中,同时该基因能够被NaCl和ABA诱导表达上调,分别在处理12和6 h时达到峰值。综上所述, KoNAC25参与了秋茄盐胁迫响应的相关调控,该结果为进一步研究秋茄KoNAC25基因在盐胁迫下的功能及其表达调控机理提供了参考。 |
| 关键词: 秋茄，NAC25基因，基因克隆，亚细胞定位，表达模式 |
| DOI：10.11931/guihaia.gxzw202408007 |
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| 基金项目:台州市科技计划项目(22nya05); 浙江省自然资源厅科技项目(2021-43); 浙江省基础公益研究计划项目(GN21C160013); 浙江省省级地质专项资金项目([省资]2024010)。第一作者：杨党(1983—), 助理工程师, 主要从事森林培育研究, (E-mail) 1798847892@qq. com。*通信作者: 杜照奎, 博士, 副教授, 硕士生导师, 主要从事红树林生态学研究, (E-mail) dzk@tzc.edu.cn。 |
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| Cloning, subcellular localization and expression analysis of the transcription factor gene KoNAC25 in Kandelia obovata |
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YANG Dang1,2, JIANG Wenjun1, WANG Yongfeng3, WANG Jianqiang4, DONG Chao4, DU Zhaokui1,2*
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1. School of Life Sciences, Taizhou University, Taizhou 318000, Zhejiang, China; 2. Zhejiang Provincial Key Laboratory of Plant Evolutionary Ecology and Conservation, Taizhou University, Taizhou 318000, Zhejiang, China; 3. Taizhou Circular Economy Development Co., Ltd., Taizhou 318000, Zhejiang, China; 4. Zhejiang Institute of Hydrogeology and Engineering Geology, Ningbo 315000, Zhejiang, China
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| Abstract: |
| NAC is a plant-specific transcription factor that plays an important role in regulating plant growth and development, hormone signaling and response to adversity stress. In our previous work, a low-temperature-induced NAC gene, KoNAC25 (GenBank accession number PP860407), was identified from the genome of the mangrove Kandelia obovata. To investigate whether the KoNAC25 gene of K. obovata is involved in the response to salt stress conditions, this study cloned the open reading frame (ORF) of the KoNAC25 gene from the cDNA of K. obovata leaves by RT-PCR, and performed bioinformatics, subcellular localization, and gene expression analyses. The results showed that: (1) the coding region of the KoNAC25 gene was 858 bp in length, encoding 285 amino acids, with a molecular weight of 32.9 kDa and a theoretical isoelectric point of 8.53. It is a hydrophilic protein, and contains none signal peptide nor transmembrane structure. (2) The N-terminus of KoNAC25 protein has a NAM superfamily structural domain, which consists of five conserved sub-structural domains and belongs to the NAM subfamily. The phylogenetic tree showed that the KoNAC25 transcription factor of K. obovata is similar to the transcription factors of Ricinus communis, Manihot esculenta and Hevea brasiliensis in the family of Euphorbiaceae. (3) The subcellular localization test confirmed that the protein was localized in the nucleus. qRT-PCR analysis showed that the expression of KoNAC25 gene was significantly higher in leaves than in flowers, and could be induced to up-regulate the expression by NaCl and ABA, which reached the peak at 12 and 6 h of treatment, respectively. In summary, KoNAC25 is involved in the regulation of salt stress response in K. obovata, and the results provide a reference for further research on the function of the KoNAC25 gene and its expression regulation mechanism under salt stress in K. obovata. |
| Key words: Kandelia obovata NAC25 gene gene cloning subcellular localization expression pattern |
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