马缨杜鹃RdNAC72基因的克隆、亚细胞定位及表达分析

Cloning, subcellular localization, and expression analysis of the RdNAC72 gene in Rhododendron delavayi

关键词:  马缨杜鹃, RdNAC72, 基因克隆, 亚细胞定位, 表达分析, 高温胁迫

Key words:  Rhododendron delavayi, RdNAC72, gene cloning, subcellular localization, expression analysis, high temperature stress

摘要:

NAC转录因子在植物生长发育和逆境胁迫中具有重要作用。然而,马缨杜鹃RdNAC72基因是否参与调控高温胁迫响应的分子机制尚未见报道。为研究RdNAC72基因与高温胁迫的相关性及其潜在的调控机制,该研究对马缨杜鹃RdNAC72基因CDS序列进行引物设计,通过PCR技术克隆RdNAC72基因全长编码序列,通过生物信息学方法对基因的结构、功能和蛋白理化性质等进行分析和预测,并利用实时荧光定量PCR(RT-qPCR)对该基因在高温胁迫下的时空表达特征及在脱落酸(ABA)胁迫下的表达模式进行分析。结果表明:(1)该基因全长1 005 bp,编码334个氨基酸,相对分子量为37.415 kDa。亚细胞定位显示RdNAC72蛋白定位于细胞核。(2)多序列比对及系统发育分析表明,RdNAC72与圆叶杜鹃的RwNAC72亲缘关系最近,顺式作用元件分析发现该基因启动子区具有参与激素、光响应、厌氧响应、低温和热应激响应元件。(3)高温胁迫可诱导RdNAC72表达且具有时空表达特异性。高温胁迫处理3 d时叶片中的RdNAC72相对表达量显著上调了31.16倍,但根和茎中该基因没有显著变化; 高温胁迫处理6 d时,根、茎、叶中的RdNAC72相对表达量均显著上调。其中,叶中的最高,显著上调了61.56倍; 其次是茎中,显著上调了50.14倍; 根中则显著上调了17.42倍。此外,还发现ABA可诱导RdNAC72表达。(4)RT-qPCR结果显示,RdHSP17.2与RdNAC72基因有协同的表达模式,并且RdHSP17.2启动子区含有多个NAC识别基序CATGTG和核心结合序列CACG,推测RdHSP17.2可能是RdNAC72转录因子的下游靶基因。该研究丰富了逆境胁迫下NAC转录因子的生物学功能,并为今后该植物的遗传育种提供了依据。

Abstract:

NAC transcription factors play important roles in plant growth, development, and stress responses. However, the molecular mechanism of the RdNAC72 gene in Rhododendron delavayi involved in response to stress remains unclear. To investigate the the roles of the RdNAC72 gene in high temperature stress response and its potential regulation mechenism, we first designed primers for cloning the full length coding sequence of the RdNAC 72 gene using PCR technology. Subsequently, the gene's structure, function, and physicochemical properties were analyzed and predicted using bioinformatics method. The spatial and temporal expression characteristics of the RdNAC72 gene under heat stress and expression pattern under ABA stress were analyzed using quantitative real-time PCR(RT-qPCR). The results were as follows:(1)The RdNAC72 gene had a full length of 1 005 bp, encoding 334 amino acids with a relative molecular weight of 37.415 kDa. Subcellular localization analysis showed that the RdNAC72 protein was located in the nucleus.(2)Multiple sequence alignment and phylogenetic analysis indicated that the RdNAC72 was most closely related to the RdNAC72 in R. williamsianum. Additionally, cis-acting element analysis revealed that the gene contained elements associated with hormone response, light response, anaerobic response, low temperature response, and heat stress response.(3)High temperature stress could induce the expression of RdNAC72, exhibiting temporal and spatial expression specificity. After three days of high temperature stress treatment, the relative expression level of the RdNAC72 gene in leaves was significantly upregulated by 31.16-fold; while no significant change was observed in roots and stems. After six days of high temperature stress treatment, the relative expression levels of RdNAC72 roots, stems, and leaves were all significantly upregulated, with the highest observed in leaves(61.56-fold), followed by stems(50.14-fold), and roots(17.42-fold). Additionally, it was found that ABA could induce the expression of RdNAC72.(4)RT-qPCR analysis demonstrated a coordinated expression pattern between RdHSP17.2 and RdNAC72 with RdHSP17.2 containing multiple NAC recognition motifs(CATGTG)and core binding sequences(CACG)in its promoter region, indicating that RdNAC72 may be a downstream target gene of RdNAC72. Therefore, the RdNAC72, a transcription factor, localized in the nucleus, responds significantly to high temperature and ABA, potentially activating the RdHSP17.2 expression to confer heat resistance. This study enriches the biological functions of NAC transcription factors, and provides the reference for the future genetic and breeding of this plant.