Molecular cloning and characterization of a DFR from developing seeds of blue-grained wheat in anthocyanin biosynthetic pathway

Document Type

Article

Publication Date

11-1-2003

Abstract

Blue-grained wheat derived from the hybrid Triticum aestivum L. × Thinopyrum ponticum (Podp.) Barkworth et D. R. Dewey (Agropyron elongatum (Host) P. Beauv., 2n=70). The molecular biological mechanism of the biosynthetic pathway of blue pigments in the blue grain remains unclear yet. Dihydroflavonol 4-reductase (DFR) is one of the key enzymes controlling flavonoid synthesis in anthocyanin biosynthetic pathway, and may directly participate in the formation of blue pigment in the aleurone layer of blue-grained wheat. Here we cloned a DFRcDNA (TaDFR) from the developing seeds of blue-grained wheat, and four DFR genomic DNAs from Th. ponticum (ThpDFRt), blue-grained wheat (TaDFRbg), white-grained offspring of light blue-grained wheat (TaDFRwg) and Chinese Spring (2n=42)(TaDFR.csg), respectively. TaDFRcDNA encodes a 354 amino-acids polypeptide with high identity to DFR from Hordeum vulgare L. (94%), Oryza sativa L. (83%). Zea mays L(84%). The result of cluster analysis showed that TaDFR cDNA nucleotide sequence has 100% identity with that of TaDFRcsg. The four DFR genomic DNAs have extraordinary high homology and each has three introns. The differences of the four DFR genomic DNAs mainly exist in introns. Southern blotting analysis showed that there are at least 3-5 DFR copies in wheat, the copy numbers in different color grain wheats are not significantly different. The hybridization band patterns were the same, but different from that of Th. ponticum. DFR in blue-grained wheat belongs to a DFR superfamily. Northern blotting analysis indicated that the DFR expressed in the developing seeds of both blue- and white-grained wheat at 15 d after flowering (DAF), the mRNA levels of DFR reached the highest at 18 DAF, then declined quickly and disappeared at 33 DAF. But the expression levels in blue-grained seeds were higher than that in white grain at the same seed developing stages. DFR transcript accumulated in young leaves, and leaf sheaths of blue- and white-grained wheat and Th. ponticum, but not detected in roots from different color wheats and developing seeds of Th. ponticum. Results indicated that there may exist some regulatory gene(s) which can increase the expression of DFR in the aleurone layer of bluegrained wheat, and thus resulting in the formation of blue pigments.

Publication Source (Journal or Book title)

Acta Botanica Sinica

First Page

1329

Last Page

1338

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