作物学报 ›› 2023, Vol. 49 ›› Issue (1): 46-61.doi: 10.3724/SP.J.1006.2023.24005
王恒波(), 张畅(), 吴明星, 李湘, 蒋钟莉, 林容潇, 郭晋隆, 阙友雄()
WANG Heng-Bo(), ZHANG Chang(), WU Ming-Xing, LI Xiang, JIANG Zhong-Li, LIN Rong-Xiao, GUO Jin-Long, QUE You-Xiong()
摘要:
NAC (NAM, ATAF和CUC)是陆生植物特有的转录因子家族, 包含18个亚家族, 其中ATAF亚家族成员广泛参与生物和非生物胁迫应答过程。本研究基于甘蔗割手密种基因组数据和栽培品种ROC22的cDNA文库, 首先, 通过比较基因组学方法, 对ATAF亚家族成员进行鉴定、蛋白多序列比对、系统进化分析及启动子区域顺式作用元件预测; 其次, 从甘蔗栽培品种克隆获得割手密种ATAF亚家族成员SsNAC2的同源基因ScNAC2, 在生物信息学分析基础上, 采用qRT-PCR技术分析该基因的组织特异性表达模式, 及其在不同外源胁迫下的表达特性; 最后, 对ScNAC2基因的编码蛋白进行亚细胞定位和转录激活活性试验。结果表明, 一共鉴定到6个ATAF亚家族成员, 开放读阅读框在889~1017 bp之间, 相对分子量介于32.067~35.819 kD之间, 理论等电点分布在5.09~8.92之间, 所有成员的编码蛋白被预测均定位于细胞核上。这些基因的Ka/Ks比值均小于1, 表明纯化选择在进化过程中起重要作用。蛋白的氨基酸序列比对显示, ATAF亚家族成员都含NAM保守结构域(由I、II、III、IV和V亚结构域组成)。系统进化分析揭示, 禾本科的甘蔗、高粱、玉米与水稻亚家族成员都聚类在一起, 表明有较近的亲缘关系, 同时拟南芥、水稻、玉米和高粱等40个ATAF亚家族成员分为两个组(Group A和Group B), 其中玉米ATAF亚家族发生明显的基因扩增现象。此外, ATAF亚家族成员启动子区域均包含响应低温、干旱和激素等逆境胁迫的顺式作用元件, 推测其参与多种生物和非生物胁迫的应答过程。进一步, 从甘蔗栽培品种ROC22中克隆获得ScNAC2基因的cDNA全长序列(GenBank登录号为OL982539), 其开放读码框为891 bp, 编码296个氨基酸残基; 该基因的编码蛋白与割手密种ATAF亚家族Group B 中SsNAC2蛋白的氨基酸序列相似性为97.99%。qRT-PCR表达分析结果表明, ScNAC2基因在甘蔗不同组织中组成型表达, 在蔗叶和蔗皮中表达量高于蔗肉、蔗芽和蔗根; 在水杨酸和茉莉酸甲酯胁迫下, 表达量显著下调; 在脱落酸、4℃低温和氯化钠胁迫下, ScNAC2基因的表达呈现由低到高的模式, 且差异达显著水平。亚细胞定位结果显示, ScNAC2-GFP融合蛋白定位在细胞核上。转录激活活性试验表明, ScNAC2蛋白不具有转录自激活活性。以上结果为深入解析甘蔗NAC-ATAF亚家族成员在响应生物和非生物胁迫应答中的生物学功能奠定了基础, 为甘蔗抗性分子育种提供潜在的基因资源。
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