芥菜型油菜4-二氢黄酮醇还原酶基因的克隆和表达分析

利用同源克隆方法, 在芥菜型油菜中克隆了DFR基因。在DNA和cDNA中扩增的DFR基因大小分别为 1 612 bp和1 214 bp。该基因含有5个内含子, 开放阅读框为1 158 bp, 预计编码385个氨基酸, 预测分子量为42 886.0 Da, 推测的等电点为5.54。DFR基因在芥菜型油菜紫叶芥和黑籽近等基因系的叶片、胚和种皮中都表达, 在四川黄籽中只在叶片和胚中表达。DFR基因在四川黄籽种皮中不表达, 导致种皮中花色素和原花色素不能合成, 从而种皮透明, 形成黄籽, 因此DFR基因是油菜种皮颜色形成途径中一个关键基因。本研究为利用该基因与种子、种皮特异启动子构建反义表达载体或RNAi载体, 阐明油菜种皮颜色形成的分子机理和创造黄籽油菜新种质奠定了基础。

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

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.