甘蓝型油菜转录因子MYB5的克隆、亚细胞定位及表达分析

Cloning, subcellular localization and expression analysis of transcription factor MYB5 in rapeseed(Brassica napus)

关键词:  甘蓝型油菜, MYB5转录因子, 基因克隆, 亚细胞定位, 镉胁迫, 表达分析

Key words:  rapeseed(Brassica napus), MYB5 transcription factor, gene cloning, subcellular localization, cadmium stress, expression analysis

摘要:

该研究为揭示甘蓝型油菜(Brassica napus)MYB4转录因子在镉(Cd)胁迫响应中的作用,以高镉积累品种‘南油868'( 2n=4x=38, AACC)为材料,克隆获得一个MYB4基因,命名为BnaMYB4,并综合运用生物信息学分析、亚细胞定位及qRT-PCR技术,对其序列特征、蛋白性质及镉胁迫下的表达模式进行分析。结果表明:(1)BnaMYB4开放阅读框全长885 bp, 编码294个氨基酸; 生物信息学分析显示其编码蛋白包含典型R2R3-MYB结构域,属于R2R3-MYB亚家族且被预测定位于细胞核。(2)多序列比对表明,BnaMYB4与甘蓝型油菜、拟南芥等物种的MYB4同源蛋白相似度较高(87.41%～98.97%); 启动子序列分析发现多个与逆境响应相关的顺式作用元件。(3)亚细胞定位实验证实BnaMYB4定位于细胞核。(4)组织表达分析显示,BnaMYB4在根、茎、叶中均有表达,其中根部表达量最高; Cd胁迫下,其在叶片中的表达量随胁迫浓度增加而轻微上升,在根部则呈轻微下降趋势。综上,BnaMYB4可能参与甘蓝型油菜对Cd胁迫的转录调控响应。该研究结果为深入解析MYB4在重金属胁迫中的功能机制及油菜抗逆育种提供了理论依据。

Abstract:

This study aims to investigate the role of the MYB4 transcription factor in cadmium(Cd)stress response in rapeseed(Brassica napus). Using the high-Cd-accumulating cultivar ‘Nanyou 868'(2n=4x=38, AACC)as experimental material, a MYB4 gene was cloned and named BnaMYB4. Bioinformatics analysis, subcellular localization, and qRT-PCR were comprehensively employed to investigate its sequence characteristics, protein properties, and expression patterns under Cd stress. The results were as follows:(1)The open reading frame of BnaMYB4 was 885 bp in length, encoding 294 amino acids; bioinformatics analysis indicated that the encoded protein contained a typical R2R3-MYB domain, belonging to the R2R3-MYB subfamily, and was predicted to localize in the nucleus.(2)Multiple sequence alignment revealed that BnaMYB4 shared high similarity(87.41%-98.97%)with MYB4 homologous proteins from species such as Brassica napus and Arabidopsis thaliana; promoter sequence analysis identified multiple stress-responsive cis-acting elements.(3)Subcellular localization experiments confirmed that BnaMYB4 localized in the nucleus.(4)Tissue expression analysis showed that BnaMYB4 was expressed in root, stem, and leaf, with the highest expression in root; under Cd stress, its expression in leaves slightly increased with increasing Cd concentrations, but decreased slightly in root. In conclusion, BnaMYB4 may be involved in the transcriptional regulatory response of rapeseed to Cd stress. This study provides a theoretical foundation for further elucidating the functional mechanism of MYB4 in heavy metal stress and for stress-resistance breeding in rapeseed.