紫花苜蓿NAC类转录因子基因MsNAC2的克隆及其功能分析

doi:10.3864/j.issn.0578-1752.2015.15.003

摘要/Abstract

摘要： 【目的】NAC转录因子是植物特有的一类转录因子，N端含有一段高度保守、约150个氨基酸组成的NAC结构域，而C端为高度变异的转录调控区。NAC转录因子不仅参与植物生长发育的调控，而且在植物抗逆反应中具有重要的调控作用。作者从紫花苜蓿中克隆了一个NAC类转录因子基因MsNAC2，期望通过分析其DNA和氨基酸序列特征，阐明其在紫花苜蓿中响应非生物胁迫的表达模式，通过在烟草中过量表达鉴定其生物学功能，为进一步了解MsNAC2在紫花苜蓿中的耐逆调控机理提供试验基础，并为通过转基因技术改善紫花苜蓿抗逆力和提高其品质奠定研究基础。【方法】应用RT-PCR和RACE技术获得紫花苜蓿MsNAC2全长序列，并进行生物信息学分析。应用real-time PCR技术分析该基因在非生物胁迫下的时空表达特征。构建MsNAC2-GFP融合表达载体，进行基因表达的亚细胞定位分析。同时构建pBI121-MsNAC2植物超表达载体，通过农杆菌介导法转化烟草叶盘，比较逆境胁迫条件下野生型烟草和转基因株系的表型和生理指标，鉴定超表达MsNAC2对烟草耐逆能力的调控效应。【结果】MsNAC2全长1 358 bp，开放阅读框长度为1 023 bp，编码340个氨基酸，编码蛋白质分子量为39.4 kD，其N端含有典型的NAC保守结构域，C端高度变异。进化树聚类分析表明，该基因与脐橙CsNAC亲缘关系较近，属于NAC蛋白的ATAF亚家族。洋葱亚细胞定位分析表明MsNAC2定位于细胞核。转录水平表达分析表明MsNAC2受250 mmol·L^-1 NaCl、20% PEG6000、0.1 mmol·L^-1 ABA和4℃胁迫诱导而显著升高，并且MsNAC2在根中的表达量要明显高于在叶中的表达量。抗性试验结果显示，在NaCl、PEG和4℃冷害胁迫下，转基因烟草苗高、根长、鲜重和干重等生长指标均高于野生型。生理指标检测结果表明，在250 mmol·L^-1 NaCl、20% PEG6000和4℃处理24 h后，转基因烟草叶片丙二醛含量明显低于野生型烟草，分别为野生型的82.6%、73.2%和77.8%。脯氨酸含量高于野生型烟草，分别达1.52倍、1.72倍和2.24倍，且SOD和POD的活性均高于野生型烟草，分别为野生型的1.101倍、1.105倍、1.33倍和1.12倍、1.08倍及1.19倍。【结论】从紫花苜蓿中克隆了一个新的NAC转录因子基因MsNAC2，该基因能够对盐、冷害和干旱胁迫产生响应，与野生型烟草相比，过量表达MsNAC₂烟草具有较强的耐盐、抗旱和抵御寒冷的能力，说明该基因可能参与调控非生物逆境胁迫的生理响应。

关键词: 紫花苜蓿, MsNAC2, 基因克隆, 功能分析

Abstract: 【Objective】NAC transcription factors belong to a unique class of transcription factors in plants. The common characteristics of the NAC proteins are the presence of a conserved NAC domain, comprising about 150 amino acids in N-terminals and a highly variable transcriptional regulation region in C-terminals. Extensive studies have revealed that NAC transcription factors not only play important roles in plant growth and development, but also have functions in regulation of responses to biotic and abiotic stresses. The objective of this study is to analyze the sequence feature and expression patterns of MsNAC2 gene with NAC transcription factor from alfalfa (Medicago sativa L.) and to illustrate the stress-resistance regulation mechanism of alfalfa through over-expression in tobacco, and through transgenic technology to improve the resistance of alfalfa and lay a foundation for the research on improving the quality.【Method】 The full length cDNA of MsNAC2 gene was cloned by RT-PCR and RACE, whose nucleotides and amino acid sequence were analyzed by bioinformatics software. The relative quantitative expression of MsNAC2 was detected by real-time PCR technology. A fusion expression vector MsNAC2-GFP was constructed to identify the subcellular localization. And a plant over-expression vector pBI121-MsNAC2 was constructed and used to transform tobacco by agrobacterium-mediated method. The phenotypic and physiological performance of the transformants were characterized under various abiotic stresses to investigate the function of MsNAC2 on stress resistance in tobacco. 【Result】 The full-length cDNA of MsNAC2 was 1 358 bp and the open reading frame was 1 023 bp, which encoded MsNAC2 comprising 340 amino acids with a molecular weight of 39.4 kD. The N-terminal amino acid residue of MsNAC2 contained a typical NAC conserved domain, and the C-terminal amino acid residue was highly variable. Phylogenetic analysis demonstrated that MsNAC2 shared high homology with CsNAC (Citrus sinensis Osbeck), and both belong to ATAF subfamily of NAC family. Subcellular localization analysis revealed that MsNAC2 was expressed in the nucleus. Transcription and expression analysis by Real-time PCR showed that MsNAC2 was up-regulated by 250 mmol·L^-1 NaCl, 20% PEG6000, 0.1 mmol·L^-1 ABA and 4℃, and the expression of MsNAC2 in root was apparently higher than those in leaf. The seedling height, root length, fresh weight and dry weight and other growth indicators of the transgenic tobacco were higher than that of the wild type in the treatments of NaCl, PEG6000, and 4℃. The physiological analysis revealed that malonaldehyde (MDA) was lower in the transformants than that in the WT, 82.60%, 73.20% and 77.80% in the treatments of NaCl, PEG6000, and 4℃, while the concentrations of proline, SOD and POD were increased. 【Conclusion】A new NAC transcription factor MsNAC2 was cloned from alfalfa, which responded to cold, drought and ABA. And MsNAC2-over- expressed tobacco possessed better anti-salt, anti-drought and anti-cold activities than wild-type tobacco. Those showed that the MsNAC2 might play an important role in stress response which indicated that this gene might participate in regulation of plant abiotic stress response.

Key words: alfalfa, MsNAC2, gene clone, function analysis

申玉华，徐振军，唐立红，杨晓坡，黄文婕，武小斌，张文波. 紫花苜蓿NAC类转录因子基因MsNAC2的克隆及其功能分析[J]. 中国农业科学, 2015, 48(15): 2925-2938.

SHEN Yu-hua, XU Zhen-jun, TANG Li-hong, YANG Xiao-po, HUANG Wen-jie, WU Xiao-bin, ZHANG Wen-bo. Cloning and Function Analysis of the MsNAC2 Gene with NAC Transcription Factor from Alfalfa[J]. Scientia Agricultura Sinica, 2015, 48(15): 2925-2938.

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