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

【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.