紫花苜蓿NAC类转录因子基因MsNAC2的克隆及其功能分析  

Cloning and Function Analysis of the Ms NAC2 Gene with NAC Transcription Factor from Alfalfa

作　　者：申玉华[1] 徐振军[1] 唐立红[1] 杨晓坡[1] 黄文婕[1] 武小斌 张文波[1] 

机构地区：[1]赤峰学院生命科学学院,内蒙古赤峰024000

出　　处：《中国农业科学》2015年第15期2925-2938,共14页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31360572)

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Ms NAC2,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Ms NAC2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Ms NAC2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Ms NAC2-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p BI121-Ms NAC2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Ms NAC2对烟草耐逆能力的调控效应。【结果】Ms NAC2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 k D,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Cs NAC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Ms NAC2定位于细胞核。转录水平表达分析表明Ms NAC2受250 mmol·L-1Na Cl、20%PEG6000、0.1 mmol·L-1 ABA和4℃胁迫诱导而显著升高,并且Ms NAC2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Na Cl、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 Na Cl、20%PEG6000和4℃处理24 h后,转基因烟草叶片丙二醛含量明显低【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 andabiotic stresses. The objective of this study is to analyze the sequence feature and expression patterns of Ms NAC2 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 c DNA of MsN AC2 gene was cloned by RT-PCR and RACE, whose nucleotides and amino acid sequence were analyzed by bioinformatics software. The relative quantitative expression of Ms NAC2 was detected by real-time PCR technology. A fusion expression vector Ms NAC2-GFP was constructed to identify the subcellular localization. And a plant over-expression vector p BI121-Ms NAC2 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 Ms NAC2 on stress resistance in tobacco.【Result】 The full-length c DNA of Ms NAC2 was 1 358 bp and the open reading frame was 1 023 bp, which encoded Ms NAC2 comprising 340 amino acids with a molecular weight of 39.4 k D. The N-terminal amino acid residue of Ms NAC2 contained a typical NAC conserved domain, and the C-terminal amino acid residue was highly variable. Phylogenetic analysis demonstrated that MsN AC2 shared high homology with Cs NAC（Citrus sinensis Osbeck）, an

关 键 词：紫花苜蓿 MS NAC2 基因克隆 功能分析 

分 类 号：S541.9[农业科学—作物学]