段迎 杨晓琳 蔡苏云 贺润丽 尹桂芳 王艳青 卢文洁 孙道旺 王莉花

摘要：【目的】克隆苦荞肉桂醇脱氢酶（CAD）基因并分析其组织表达特性，为深入研究CAD基因在苦荞果壳形成中的分子调控机制提供理论依据。【方法】基于苦荞转录组测序结果，从苦荞厚果壳品种云荞1号和薄果壳品种小米荞克隆CAD基因，对其序列进行生物信息学分析，并利用实时荧光定量PCR（qRT-PCR）检测CAD基因在不同果壳厚度类型（厚果壳苦荞和薄果壳苦荞）不同组织的表达情况。【结果】从薄果壳苦荞和厚果壳苦荞中均克隆获得2条苦荞CAD基因，且这2条基因序列在薄果壳苦荞与厚果壳苦荞中均完全一致，命名为FtCAD-1和FtCAD-2。FtCAD-1基因的开放阅读框（ORF）长度为876 bp，编码291个氨基酸残基，为疏水性的稳定酸性蛋白，定位于细胞核和细胞质;FtCAD-2基因的ORF长度为1083 bp，編码360个氨基酸残基，为亲水性的稳定酸性蛋白，定位于细胞质。FtCAD-1和FtCAD-2蛋白均具有CAD蛋白3个典型的保守结构域，且不具有跨膜结构域和信号肽，属于非分泌蛋白。FtCAD-1与数据库目标蛋白2cf5.1.B的结构相似度为74.74%，而FtCAD-2与数据库目标蛋白5z0c.1.A的结构相似度为63.03%。FtCAD-1与拟南芥的第一类CAD蛋白（AtCAD4和AtCAD5）的亲缘关系较近;FtCAD-2与拟南芥的第二类CAD蛋白（AtCAD2、AtCAD3、AtCAD6等）的亲缘关系较近。FtCAD-1和FtCAD-2基因均在种仁中的相对表达量最高，且厚果壳苦荞与薄果壳苦荞间无显著差异（P>0.05）。FtCAD-1基因在厚果壳苦荞叶、花和果壳中的相对表达量显著（P<0.05，下同）或极显著（P<0.01）高于薄果壳苦荞，尤其是在厚果壳苦荞果壳中相对表达量是薄果壳苦荞的16倍。FtCAD-2基因除了在薄果壳苦荞果壳中相对表达量显著高于厚果壳苦荞外，在其他组织中的相对表达量均表现为厚果壳苦荞高于薄果壳苦荞。【结论】FtCAD-1属于第一类CAD基因，具有明显的组织表达特异性，推测其在苦荞木质素生物合成过程中发挥重要调控作用。

关键词： 苦荞;肉桂醇脱氢酶（CAD）;RT-PCR克隆;生物信息学分析;实时荧光定量PCR

中图分类号： S517.035.3                             文献标志码： A 文章编号：2095-1191（2021）12-3340-10

Cloning and tissue expression analysis of FtCAD-1 and FtCAD-2 genes from tartary buckwheat

DUAN Ying1， YANG Xiao-lin1， CAI Su-yun1， HE Run-li1*， YIN Gui-fang2，

WANG Yan-qing2， LU Wen-jie2， SUN Dao-wang2， WANG Li-hua2*

（1School of Traditional Chinese Medicine and Food Engineering，Shanxi University of Traditional Chinese Medicine，Taiyuan  030619， China; 2Yunnan Academy of Agricultural Sciences， Biotechnology and Genetic Germplasm Resources Research Institutes/Yunnan Provincial Key Laboratory of Agricultural Biotechnology/Key Laboratory of Southwestern Crop Gene Resources and Germplasm Innovation，Ministry of Agriculture and Rural Affairs， Kunming  650201， China）

Abstract：【Objective】To clone the cinnamyl alcohol dehydrogenase（CAD） gene of tartary buckwheat and analyze its tissue expression characteristics， in order to provide a theoretical reference for further study of the molecular mechanism underlying CAD gene regulation during the formation of the thin husk of tartary buckwheat. 【Method】The CAD gene of tartary buckwheat with thin husk （Yunqiao 1） and tartary buckwheat with thick husk （rice buckwheat） was cloned based on the related fragments of tartary buckwheat transcriptome and analyzed by bioinformatics. The expression of CAD in different tissues of the different husk types（thin husk and thick husk） was studied by real-time fluorescence quantitative PCR. 【Result】Two CAD genes of tartary buckwheat were cloned from tartary buckwheat with thin and thick husks，and found to be completely identical to FtCAD-1 and FtCAD-2. The open reading frame（ORF） of FtCAD-1 gene was 876 bp in length and encoded 291 amino acid residues. It was predicted to be a hydrophobic stable acidic protein and was located in the nucleus and cytoplasm. The ORF of FtCAD-2 gene was 1083 bp in length and encoded 360 amino acid residues predicted to be a hydrophilic and stable acidic protein located in the cytoplasm. Both proteins had three typical conserved domains of CAD proteins，but did not have transmembrane domains or signal peptides， and so belong to non-secreted proteins. The structural similarity between FtCAD-1 and the database target protein 2cf5.1.B was 74.74%， while the structural similarity between FtCAD-2 and the database target protein 5z0c.1.A was 63.03%. FtCAD-1 was closely related to the first type of protein （AtCAD4， AtCAD5） in Arabidopsis lignin synthesis， but FtCAD-2 was closely related to the second type of CAD protein （AtCAD2， AtCAD3， AtCAD6 et al.） in Arabidopsis. The relative expression levels of FtCAD-1 and FtCAD-2 genes were the highest in the seed kernels， where there was no significant difference observed between the thick-husk and the thin-husk tartary buckwheat types（P>0.05）. The relative expression of FtCAD-1 gene in the leaves，flowers and husks of tartary buckwheat with thick husks was significantly （P<0.05， the same below） or very significantly （P<0.01） higher than those in tartary buckwheat with thin husks. This was most notable in the husks，where the relative expression level of FtCAD-1 thick husked tartary buckwheat was 16 times higher than that in tartary buckwheat with thin husk. In contrast，the relative expression level of the FtCAD-2 gene was significantly higher in thin husks than that in thick husks， while in other tissues FtCAD-1 showed a higher relative expression level in thick husked tartary buckwheat than that in thin husked tartary buckwheat. 【Conclusion】The FtCAD-1 gene belongs to the first type of CAD gene， it has tissue expression specificityand plays an important role in the regulation of lignin biosynthesis in tartary buckwheat.

Key words：tartary buckwheat; cinnamyl alcohol dehydrogenase（CAD）; RT-PCR cloning; bioinformatics analysis; real-time fluorescence quantitative PCR

Foundation item： Construction Project National Technical System of Oat and Buckwheat Industry（CARS-07-C-2）;Key Research and Development Project of Shanxi（201803D221012-6）; Shanxi Province Natural Science Research General Project（20210302123231）

0 引言

【研究意义】苦荞[Fagopyrum tataricum（L.） Gaertn]具有利耳目、续精神、益气力等功效，是我国药食同源文化的典型体现（王世霞等，2016;周良等，2019）。苦荞……