摘要：

WRKY转录因子家族在调控非生物胁迫中具有重要的作用。为系统地分析烟草NtWRKY11基因的序列特征、表达模式,探究该基因在低温和干旱等非生物胁迫下的反应机制,以普通烟草cDNA为模板进行 PCR 扩增,克隆NtWRKY11基因的全长cDNA序列,利用生物信息学软件分析NtWRKY11蛋白的基本性质,构建植物表达载体研究其亚细胞定位,利用qRT-PCR技术检测烟草盛花期不同组织以及不同胁迫下NtWRKY11基因的表达情况。结果表明,烟草NtWRKY11基因cDNA全长999 bp,编码332个氨基酸,与苎麻BnWRKY11的相似性达54.23%。其启动子含有1个MBS、1个MYB和3个ARBE顺式作用元件,这3种元件可能共同发挥作用,使植物的抗旱性增强;还含有3个水杨酸响应的顺式作用元件TCA-element,能够提高植物的耐低温性。亚细胞定位结果表明,NtWRKY11蛋白定位在细胞核上。盛花期不同组织的表达分析表明,NtWRKY11在老叶中高表达,极显著高于根中的表达量,而花中的表达量极显著低于根中,茎和新叶中的表达量均与根中差异不显著。非生物胁迫下的表达分析表明,该基因在低温、干旱胁迫下的相对表达量极显著高于正常(CK),在高盐胁迫处理下的相对表达量与CK无显著差异,而在高温胁迫下的相对表达量极显著低于CK。综上所述,NtWRKY11在老叶中高表达,在干旱胁迫、低温胁迫下表达量增加,表明该基因作为正向转录因子调控了干旱和低温胁迫。

关键词: 烟草, NtWRKY11, 干旱, 低温, 转录因子, 非生物胁迫

Abstract:

The WRKY transcription factor family plays an important role in regulating abiotic stress.To systematically analyze the sequence characteristics and expression patterns of NtWRKY11 gene in tobacco,and explore the response mechanism of NtWRKY11 under abiotic stress such as low temperature and drought,the full-length cDNA sequence of NtWRKY11 gene was amplified by PCR using common tobacco cDNA as a template.The basic properties of NtWRKY11 protein were analyzed by bioinformatics software and the subcellular localization of NtWRKY11 protein was studied by constructing a plant expression vector.The expression of NtWRKY11 gene was detected by qRT-PCR technology in different tissues at the full flowering stage and under different abiotic stresses.The results showed that the full-length cDNA of NtWRKY11 gene in tobacco was 999 bp,encoding 332 amino acids,and it shared 54.23% similarity with ramie BnWRKY11.Its promoter region contained three kinds of cis-acting elements(one MBS,one MYB and three ARBE),which probably worked together to enhance the drought resistance of plants.It also contained three salicylic acid-responsive cis-acting elements(TCA-element),which could improve the low temperature tolerance of plants.Subcellular localization results indicated that the NtWRKY11 protein was located in the nucleus.The expression analysis of NtWRKY11 in different tissues at the full flowering stage showed that NtWRKY11 was highly expressed in old leaves,significantly higher than in roots.However,the expression in flowers was significantly lower than that in roots,and there was no significant difference between stems,new leaves and roots. The expression analysis under abiotic stress showed that the relative expression of the gene was significantly higher than that of normal (CK).The relative expression level under high salt stress was not significantly different from CK,while the relative expression level under high temperature stress was significantly lower than CK.All in all,NtWRKY11 is highly expressed in old leaves,and its expression level is enhanced under abiotic stresses such as drought stress and low temperature stress,indicating that this gene acts as a forward transcription factor to regulate drought and low temperature stress.

Key words: Tobacco, NtWRKY11, Drought, Low temperature, Transcription factor, Abiotic stress