关键词:

芥菜型油菜, 4-二氢黄酮醇还原酶基因, 克隆, RT-PCR分析

Abstract:

The yellow-seeded cultivars of rapeseed have higher oil content than those of the black-seeded varieties. Yellow seeds of rapeseed have lower fibre content compared with those of black-seeded varieties. They may also have higher protein content than black seeds. Higher protein content and lower crude fibre content are desirable traits in rapeseed meal, which is the most important fodder product of rape. However, there is little knowledge on the biosynthesis of seed coat color in rapeseed. Therefore, it is necessary to elucidate the mechanism of seed coat color in rapeseed. Dihydroflavonol 4-reductase (DFR) gene is a key gene in the way of proanthocyanidins biosynthesis of seed coat in Arabidopsis thaliana, its mutation caused transparent testa. In order to study molecular mechanism of seed coat color in rapeseed, dihydroflavonol 4-reductase (DFR) gene was cloned from Brassica juncea using homology-based cloning strategy. The cloned gene of 1 612 bp contains 5 introns. The complementary DNA (cDNA) consists of 1 214 bp and has an open reading frame of 1 158 bp encoding a deduced polypeptide of 385 amino acids with a predicted molecular weight of 42 886.0 Da and an estimated isoelectric point of 5.54. RT-PCR analysis showed that DFR expressed in leaves, embryos and seed coats of Purple-leaf Mustard (PLM) and two black-seeded near-isogenic lines (NILs) developed from backcross breeding using Sichuan Yellow (SY) as a current parent, whereas the gene expressed only in the leaves and embryos of SY, not in the seed coats. No expression of DFR blocked the biosynthesis of anthocyanidins and proanthocyanidins in yellow seed coats, and seeds displayed yellow appearance because of transparent testa. So DFR is a key gene for the formation of seed coat color in B. juncea. This work provided a foundation for understanding 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.

Key words:

Brassica juncea, Dihydroflavonol 4-reductase gene, Cloning, RT-PCR analysis