Cloning and expression analysis of leucoanthocyanidin reductase gene in Fagopyrum dibotrys

The leucoanthocyantin reducase (LAR) gene, an important functional gene of catechins biosynthesis pathway, was cloned from Fagopyrum dibotrys (D.Don) Hara by degenerate PCR and rapid amplification of cDNA ends (RACE).  The full-length cDNA of FdLAR is 1 581 bp (GenBank accession: JN793953), containing a 1 176 bp ORF encoding a 391 amino acids protein, and its 3'-untranslated region has an obvious polyadenylation signal.  The recombinant plasmid containing FdLAR completed ORF was transformed into E. coli BL21 (DE3).  The target fusion peptide with molecular weight of 66 kD was expressed under the condition of 16 ℃ and induced by IPTG at final concentration of 1.0 mmol·L⁻¹.  Bioinformation analysis indicated that the amino acid sequence of FdLAR showed great homology to other LAR with the NADB-Rossmann conversed domain in the N-terminus.  Real-time quantitative PCR was used to detect the expression levels of FdLAR gene during different development periods.  The determination of flavonoids contents in appropriate rhizomes showed that the relationship between FdLAR gene expression and the accumulation of flavonoids displayed different trends during vegetative growth and reproductive growth stages, suggesting that the FdLAR gene may be involved in the pathway of flavonoid metabolisms in Fagopyrum dibotrys.