Cloning and Expression Analysis of Dihydroflavonol 4-Reductase Gene in Brassica juncea

Abstract

Dihydroflavonol 4-reductase ( DFR) gene is a key gene of proanthocyanidins biosynthesis pathway in seed coat of Arabidopsis thaliana. The mutation of this gene brings about a transparent testa. To study molecular mechanism of seed coat color in Brassica, DFR gene was cloned from B. juncea using homology-based clone strategy. The cloned gene 1,612 bp in length contains 5 introns. The complementary DNA (cDNA) consists of 1,214 bp and has a 1,158 bp open reading frame encoding a deduced polypeptide of 385 amino acids with a predicted molecular weight of 42,886 Da and an estimated isoelectric point of 5.54. RT-PCR analysis showed that DFR was expressed in leaves, embryos, and seed coats of Purple-Leaf Mustard and 2 black-seeded near-isogenic lines developed from backcross breeding using Sichuan Yellow as a recurrent parent. Gene DFR was expressed only in the leaves and embryos of Sichuan Yellow, but not in seed coats. No expression of DFR blocked the biosynthesis of anthocyanidins and proanthocyanidins in the yellow seed coat, and seeds appeared yellow because of transparent testa. Gene DFR is essential in the formation of seed coat color in B. juncea. This study provided a foundation for understanding the molecular mechanism of seed coat color and developing novel yellow-seeded rapeseed germplasm through antisense expression or RNAi suppression of DFR gene in black-seeded cultivars using a seed- or seed-coat-specific promoter.