Abstract: To investigate the role of the leucoanthocyanidin reductase-encoding gene SpLAR in proanthocyanidin biosynthesis in Spirodela polyrrhiza, the full-length SpLAR cDNA sequence was obtained from transcriptome data and the gene was successfully cloned. The characteristics of the SpLAR protein were predicted, and the expression levels of the SpLAR gene under different culture days and nutrient stress conditions were detected. Additionally, by measuring the accumulation of proanthocyanidins under corresponding conditions, the correlation between the expression level of the SpLAR gene and the accumulation of proanthocyanidins was further analyzed. The pCAMBIA1301-SpLAR plant expression vector was constructed using homologous recombination technology. Bioinformatics analysis results showed that the gene was 1 059 bp in length, encoding 352 amino acids, with a predicted molecular weight of 38 ku. It was a hydrophilic and unstable protein, containing one transmembrane domain, having no signal peptide, and belonging to the NADB_Rossmann superfamily. The SpLAR protein had the highest similarity with the homologous protein from Colocasia esculenta. RT -qPCR and content determination results indicated that the expression level of the SpLAR gene showed a dynamic trend of first increasing and then decreasing, and was positively correlated with the accumulation of proanthocyanidins. Under Datko treatment, the expression level of the SpLAR gene was the highest at 9 d, and at this time, the accumulation of proanthocyanidins reached a peak of 20.6 mg/g. In addition, the nutrient stress treatment could significantly promote the expression of the SpLAR gene and the biosynthesis of proanthocyanidins, increasing the accumulation to 24.8 mg/g at 9 d.

Key words: Spirodela polyrrhiza, SpLAR gene, bioinformatics, expression analysis, vector construction