秋茄KoNAC25基因的克隆、亚细胞定位及表达分析

Cloning, subcellular localization and expression analysis of KoNAC25 gene in Kandelia obovata

关键词:  秋茄, NAC25基因, 基因克隆, 亚细胞定位, 表达模式

Key words:  Kandelia obovata, NAC25 gene, gene cloning, subcellular localization, expression pattern

摘要:

NAC转录因子在植物中广泛存在且为植物所特有,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1个受低温诱导的NAC基因KoNAC25(GenBank登录号PP860407)。为探究KoNAC25基因是否参与秋茄盐胁迫响应,该研究通过 RT-PCR技术从秋茄叶片cDNA中克隆KoNAC25基因,并对其进行序列特征、系统进化、亚细胞定位及基因表达分析。结果表明:(1)秋茄KoNAC25基因的开放阅读框(ORF)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。KoNAC25为亲水性蛋白,不含信号肽且无跨膜结构。(2)序列同源比对显示,KoNAC25蛋白N端具有1个NAM超家族结构域,由5个保守的亚结构域构成,属于NAM亚家族成员。(3)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。(4)亚细胞定位试验证实,KoNAC25蛋白定位于细胞核。(5)qRT-PCR分析结果表明,KoNAC25基因在叶中表达量显著高于花中,同时该基因能够被盐(NaCl)和脱落酸(ABA)诱导表达上调,分别在处理12 h和6 h时达到峰值。综上认为,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。

Abstract:

NAC transcription factors are widely present in plants and unique to plants, which play important roles in regulating plant growth and development, hormone signaling, and the response to adverse 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 KoNAC25 gene from the cDNA of K. obovata leaves via RT-PCR and performed sequence characterization, phylogenetic evolution, subcellular localization, and gene expression analyses. The results were as follows:(1)The open reading frame(ORF)of the KoNAC25 gene was 858 bp in length and encoded 285 amino acids, with a molecular weight of 32.9 kDa and a theoretical isoelectric point of 8.53. KoNAC25 was a hydrophilic protein and contained no signal peptide or transmembrane structure, and its secondary structure was predominantly random coils(58.95%).(2)Amino acid sequence homology alignment showed the N-terminus of the KoNAC25 protein had a NAM superfamily structural domain, which consisted of five conserved substructural domains(ABCDE)and belonged to the NAM subfamily.(3)The phylogenetic tree revealed that the KoNAC25 transcription factor of K. obovata was similar to the transcription factors of Ricinus communis, Manihot esculenta, and Hevea brasiliensis.(4)The subcellular localization test confirmed that the protein was localized in the nucleus, which was consistent with bioinformatics prediction.(5)qRT-PCR analysis result revealed that the expression of the KoNAC25 gene was significantly greater in leaves than in flowers and could be induced to upregulate its expression by NaCl(200 mmol·L^-1)and abscisic acid(ABA)(100 μmol·L^-1), which peaked under 12 h treatment and 6 h treatment, respectively; however, the expression level of this gene was not induced by salicylic acid(100 μmol·L^-1)and methyl jasmonate(100 μmol·L^-1). In summary, KoNAC25 is involved in the related regulation of the salt stress response in Kandelia obovata, which provides a reference for further research on the function of KoNAC25 gene and its expression regulatory mechanism under salt stress in K. obovata.