目的 克隆掌叶大黄Rheum palmatum转录因子基因RpNAC1，进行生物信息学、亚细胞定位及表达模式分析。方法 根据转录组中一个NAC unigene，利用RT-PCR克隆开放阅读框（open reading frame，ORF）。通过生物信息软件预测基因编码蛋白的理化性质、结构域等分子特征。采用DNAStar 6.0和MEGA 7.0分别进行氨基酸序列比对和进化分析。构建绿色荧光蛋白（GFP）融合表达载体，以农杆菌侵染烟草叶片的瞬时表达法分析亚细胞定位。运用qRT-PCR检测基因表达模式。结果 分离到RpNAC1基因，ORF（1269 bp），编码一个由422个氨基酸组成的蛋白质，相对分子质量47 070，等电点5.80，包含一个保守NAC结构域（32～181），与多种植物NAC蛋白一致性较高（62.42%～88.7%），聚在植物NAC分子进化树的NAC2分支，与甜菜NAC（XP_010694507）亲缘关系较近。RpNAC1-GFP融合蛋白定位在烟草细胞核内。RpNAC1基因在一年生植株根中表达量最高，根茎中表达量最低，相对表达量分别为叶中的5.37、0.012倍；该基因响应200 μmol/L赤霉素（gibberellin，GA3）处理后在24 h内总体上调，200 μmol/L茉莉酸甲酯（methyl jasmonate，MeJA）处理1 h和12 h基因显著上调，200 μmol/L水杨酸（salicylic acid，SA）处理12 h显著诱导基因表达，200 μmol/L脱落酸（abscisic acid，ABA）处理抑制基因表达，200 μmol/L乙烯（ethylene，ET）处理未见明显变化。结论 获得掌叶大黄RpNAC1基因序列及表达特征，为进一步研究其在蒽醌类成分合成与积累中的转录调控作用提供支撑。

Objective To clone a NAC transcription factor gene RpNAC1 from Dahuang (Rheum palmatum) followed by bioinformatics, subcellular localization and expression pattern analysis. Methods According to a NAC transcription factor unigene in transcriptome, the open reading frame (ORF) was cloned by RT-PCR. The physical and chemical properties, protein structure, and other molecular characteristics of the deduced protein RpNAC1 were predicted by bioinformatics tools. DNAStar 6.0 and MEGA7.0 were used for multiple sequence alignment and phylogenetic tree analyses, respectively. Green fluorescent protein (GFP) fused expression vector was constructed and subcellular localization of RpNAC1 was observed by Agrobacterium tumefaciens transient expression method in tobacco. Quantitative PCR was employed for gene expression analyses. Results The ORF of RpNAC1 was 1269 bp in size, encoding a 422-aa protein with a molecular weight of 47 070 and an isoelectric point of 5.80. The deduced RpNAC1, containing a conserved NAC domain (32—181). RpNAC1 protein was highly consistent with other plant’s NAC proteins (62.42%—88.7%), and was clustered in NAC2 subclass of plant NAC molecular phylogenetic tree, and was closely related to Beta vulgaris NAC (XP_010694507). The RpNAC1-GFP fusion protein was located in the nucleus of tobacco mesophyll cells. The qRT-PCR analyses showed that the abundance of RpNAC1 gene was the highest in roots and the lowest in rhizomes, and the relative expression levels were 5.37 and 0.012 times higher than those in leaves of R. palmatum seedlings at one-year stage. After 200 μmol/L gibberellin (GA3) treatment, RpNAC1 transcript was up-regulated within 24 h. Methyl jasmonate (MeJA, 200 μmol/L) treatment for 1 h and 12 h significantly up-regulated its expression level. Salicylic acid (SA, 200 μmol/L) treatment also significantly induced gene expression. Whereas, Abscisic acid (ABA, 200 μmol/L) treatment inhibited gene expression and 200 μmol/L ethylene (ET) treatment did not show any effects. Conclusion The sequence and expression characteristics of RpNAC1 gene were obtained, which will provide the reference for further study on the biological function of the gene in the biosynthesis and accumulation regulation of anthraquinones in R. palmatum.

R282.12

国家自然科学基金资助项目（82104334）；陕西中医药大学校级课题（2020PG29）；陕西中医药大学新进博士科研启动经费（104080001）；陕西中医药大学学科创新团队项目（2019-QN01）；中央本级重大增减支项目（2060302）