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J Integr Plant Biol, 2003, 45 (11): 1329-1338, Research Article
Molecular Cloning and Characterization of a DFR from Developing Seeds of Blue-grained Wheat in Anthocyanin Biosynthetic Pathway
YANG Guo-Hua*, ZHAO Xue-Qiang*, LI Bin, LIU Jian-Zhong, ZHENG Qi, TONG Yi-Ping, LI Zhen-Sheng**
doi:
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 DFR cDNA (TaDFR ) from the developing seeds of blue-grained wheat, and four DFR genomic DNAs from Th. ponticum (ThpDFR.t), blue-grained wheat (TaDFR.bg), white-grained offspring of light blue-grained wheat (TaDFR.wg) and Chinese Spring (2n=42) (TaDFR.csg), respectively. TaDFR cDNA 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 ofTaDFR.csg. 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 transcripts accumu-lated 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 blue-grained wheat, and thus resulting in the formation of blue pigments.

蓝粒小麦花青素生物合成途径中的二氢黄酮醇4 -还原酶基因的分子克隆
杨国华  赵学强 李 滨 刘建中 郑 琪 童依平  李振声

(1.中国科学院遗传与发育生物学研究所植物细胞与染色体工程国家重点实验室, 北京 100101;
2.天津师范大学化学与生命科学学院,天津 300074; 3.中国科学院生态环境研究中心,北京 100085)


摘要: 蓝色色素在蓝粒小麦种子糊粉层中的生物合成途径的分子生物学机制至今仍不清楚。应用RT-PCR和RACE方法从蓝粒小麦正在发育的种子中克隆到一个编码二氢黄酮醇4-还原酶的基因(DFR)。推测其为花青素生物合成途径中的一个关键基因,且与蓝粒小麦中蓝色色素形成密切相关;其开放阅读框编码一个包含354个氨基酸残基的多肽,与一些从其他植物中已克隆到的DFR有很高的同源性:大麦(94%)、水稻(83%)、玉米(84%)。从长穗偃麦草(2n=70)、蓝粒小麦、浅蓝粒小麦自交产生的白粒后代小麦以及中国春的基因组中分别分离到一个全长DFR序列。经聚类分析表明DFR cDNA核甘酸序列与从中国春基因组中克隆的DFR具有100%的同源性,且与长穗偃麦草、蓝粒小麦、白粒小麦基因组中分离的DFR均有很高的同源性。4个DFR基因组DNA均含有3个内含子,且它们之间的差异主要在内含子区,表明该基因在进化上很保守。经Southern杂交分析,DFR在小麦中至少有3~5个拷贝,不同小麦材料间未见明显差异,但与长穗偃麦草有明显差异,属于一个DFR超基因家族。Northern分析表明该DFR在蓝粒和白粒种子的不同发育时期的表达存在明显差异,都在开花后大约18 d表达最强,在同一时期的蓝白种子中,DFR在蓝粒种子中的表达量高于白粒。DFR 转录本在小麦和长穗偃麦草的幼叶中积累多,但在芽鞘中的表达显著低于幼叶中;在小麦的根和长穗偃麦草的发育种子中均未检测到DFR的表达。推测蓝粒小麦中可能存在调控DFR在蓝粒小麦中表达的调控基因,类似于玉米花青素合成途径中的调节基因。
关键词: 蓝粒小麦;花青素生物合成途径;二氢黄酮醇4-还原酶;RACE;长穗偃麦草(2n=70)

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