白木香转录因子AsNAC2的克隆及表达分析

Molecular cloning and expression characterization of a transcription factor AsNAC2 in Aquilaria sinensis

目的 对珍稀南药沉香基原植物白木香Aquilaria sinensis As NAC2转录因子进行分子克隆,并对其进行生物信息学和表达模式分析。方法 以白木香茎的总RNA反转录的c DNA为模板,采用PCR技术获取基因编码序列(coding sequence,CDS)全长;利用生物信息学软件预测蛋白的理化性质、结构域和亚细胞定位等特性;运用DNAMAN8.0和MEGA6.0等软件分别进行多序列比对和进化关系分析;利用qRT-PCR技术检测AsNAC2基因在不同组织和伤害胁迫下的表达特性。结果克隆获得AsNAC2基因(GenBank注册号MT130201),开放阅读框(openreadingframe,ORF)全长864bp,编码1条由287个氨基酸组成的弱酸性的稳定的亲水性蛋白,该蛋白定位在细胞核中,不存在跨膜结构域。序列比对和系统进化树分析显示,As NAC2蛋白N端具有典型的NAM结构域,与锦葵目中的可可、哥伦比亚锦葵和榴莲NAC 2-like蛋白亲缘关系近,属于NAC转录因子家族中的ATAF亚组。qRT-PCR结果显示,AsNAC2基因主要在健康白木香的根和茎中表达,全断干伤害可显著诱导茎中AsNAC2基因表达量的上调,在2 h达到峰值,此后缓慢下降,至48 h基本恢复正常。结论 首次在白木香中克隆和鉴定了AsNAC2基因,该基因易受伤害诱导表达,且在伤害早期相对应。

Objective To molecular clone transcription factor of Aquilaria sinensis encoding gene AsNAC2, a rare southern medicine sedum-based plant, in order to analyze its bioinformatics and expression pattern. Methods The full length of the coding sequence(CDS) was obtained by PCR using total RNA reverse transcribed c DNA from the stems of A. sinensis as the template;Bioinformatics software was used to predict the physicochemical properties, structural domains and subcellular localization of the protein;DNAMAN8.0 and MEGA6.0 were used to perform multiple sequence alignment and evolutionary relationship analysis,respectively. The expression characteristics of As NAC2 gene under different tissues and injury stresses were examined by q RT-PCR.Results As NAC2 gene(GenBank accession MT130201) was cloned from A. sinensis;The full length of open reading frame(ORF) was 864 bp. Codification Code a weakly acidic, stable hydrophilic protein consisting of 287 amino acids, which was localized in the nucleus and no transmembrane structural domain existed. Sequence alignment and phylogenetic tree analysis showed that the N-terminal As NAC2 protein had a typical NAM structural domain and was closely related to Theobroma Cacao, Herrania umbratica and, Durio zibethinus. NAC 2-like proteins in the mallow order, belonging to the ATAF subgroup of the NAC transcription factor family. qRT-PCR results showed that As NAC2 gene was mainly expressed in the roots and stems of healthy A. sinensis, and whole-break dry injury significantly induced the up-regulation of As NAC2 gene expression in the stems, reaching a peak at 2 h, and then slowly decreasing until 48 h when it basically returned to normal. Conclusion The AsNAC2 gene was cloned and characterized from A. sinensis for the first time, which is susceptible to injury-induced expression and correspondingly early in the injury.