Cloning， expression and trans-activation activity analysis of DcNAC1 gene from Dendrobium catenatum
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摘要: 【目的】克隆铁皮石斛NAC转录因子基因（DcNAC1），并进行表达模式及转录自激活活性分析，为铁皮石斛抗逆相关基因鉴定及其分子机制研究提供参考。【方法】以铁皮石斛cDNA为模板，PCR扩增DcNAC1基因，运用生物信息学软件分析DcNAC1蛋白的理化性质、保守结构域、信号肽、跨膜结构域及亚细胞定位，通过实时荧光定量PCR检测DcNAC1基因在不同组织和不同逆境胁迫下的表达模式。同时构建该基因的酵母表达载体，分析其转录自激活活性。【结果】从铁皮石斛中PCR扩增获得DcNAC1基因的开放阅读框（ORF），全长为945 bp，与参考序列（LOC110104882）的核苷酸序列相似性为100%。该基因编码314个氨基酸残基，蛋白分子量为35.40 kD，理论等电点（pI）为8.16，为不稳定的亲水性蛋白，定位于细胞核，不含信号肽和跨膜结构域，含有特征性的NAC保守结构域。DcNAC1基因的启动子序列含茉莉酸甲酯响应元件（CGTCA-motif和TGACG-motif）、胁迫响应元件（TC-rich repeats）、光响应元件（G-box）、干旱诱导MYB结合位点（MBS）和低温响应元件（LTR）。根中DcNAC1基因的相对表达量在高温胁迫和低温胁迫处理6 h分别达最高，显著高于处理0 h（P＜0.05，下同）；茎中DcNAC1基因在盐胁迫处理48 h的相对表达量达最高，显著高于处理0 h。将构建的重组质粒pGBKT7-DcNAC1转化酵母菌株Y2HGold，结果发现该重组质粒无毒性，DcNAC1蛋白具有自激活活性。【结论】 DcNAC1基因表达受到茉莉酸、低温、干旱、光信号和逆境胁迫等多种信号的调控。DcNAC1蛋白具有自激活活性，通过激活下游基因的表达，参与到植物生长发育和逆境胁迫响应的转录调控过程中。Abstract: 【Objective】 This study was to dissect the expression pattern and trans-activation activity of the NAC gene （DcNAC1）of Dendrobium catenatum， thus providing a foundation for stress-related genes identification and elucidating the molecular mechanism of D. catenatum stress-resistance.【Method】 DcNAC1 gene was amplified by PCR using D. catenatum cDNA as template. The physical and chemical properties，conserved domain，signal peptide，transmembrane domain and subcellular location of DcNAC1 protein were analyzed by bioinformatics softwares. The expression patterns of DcNAC1 gene in different tissues and under different stresses were detected by real-time fluorescence quantitative PCR. At the same time，yeast expression vector of this gene was constructed and its transcriptional self-activation activity was analyzed.【Result】 The open reading frame（ORF）of DcNAC1 gene was obtained by PCR amplification from D. catenatum. The total length was 945 bp，and the nucleotide sequence similarity to the reference sequence（LOC110104882）was 100%. This gene encoded 314 amino acid residues，had a molecular weight of 35.40 kD and a theoretical isoelectric point （pI）of 8.16. It was an unstable hydrophilic protein，localized in the nucleus，free of signal peptides and transmembrane domains，and contained a characteristic NAC conserved domain. The promoter sequences of DcNAC1 gene included jalapic acid response elements（CGTCA-motif and TGACG-motif），stress response elements（TC-rich repeats），light response elements（G-box），drought-induced MYB binding sites（MBS）and low temperature response elements（LTR）. The relative expression of DcNAC1 gene in root reached the highest level at 6 h under high temperature stress and low temperature stress，respectively，and was significantly higher than that at 0 h under high temperature stress（P＜0.05，the same below）. The relative expression of DcNAC1 gene in stems was the highest at 48 h after salt stress treatment，which was significantly higher than that at 0 h. The recombinant plasmid pGBKT7-DcNAC1 was transformed into yeast strain Y2HGold. The results showed that the recombinant plasmid was not toxic，but DcNAC1 protein had certain self-activation activity.【Conclusion】DcNAC1 gene expression is regulated by jasmonic acid，low temperature，drought，light signal and stress. DcNAC1 protein has self-activating activity，which is involved in transcriptional regulation of plant growth and development and response to stress by activating the expression of downstream genes.
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