金荞麦二氢黄酮醇4-还原酶基因（FdDFR1）的克隆及序列分析

 【目的】克隆金荞麦二氢黄酮醇4-还原酶基因（FdDFR1），分析其序列特征。【方法】利用同源克隆的原理，采用RACE结合cDNA文库筛选的方法，从金荞麦cDNA文库中克隆DFR基因（FdDFR1）;对其序列进行生物信息学分析和Southern杂交检测。【结果】克隆到一个DFR蛋白基因（FdDFR1），通过Southern杂交分析，推测在金荞麦基因组中DFR基因是1～2个基因的小家族，FdDFR1是单拷贝基因;FdDFR1基因编码一个长341个氨基酸的蛋白质，在N端存在一个NADP结合位点“VTGASGFVGSWLVMRLLEHGY”，还存在一个底物结合特异性决定的氨基酸基序“TVNVEEKQKPVYDETCWSDVDFCRRV”。【结论】首次从金荞麦中克隆到类黄酮代谢的中期关键酶基因（FdDFR1），它具有DFR同源基因的典型特征。

Cloning and Sequence Analysis of a DFR Gene from Fagopyrum dibotrys (D.Don) Hara

【Objective】 This work was aimed to investigate the sequence characteristics of a DFR gene from Fagopyrum dibotrys (D.Don) Hara. 【Method】According to the obtained homologous probe from other plant’s DFRs, DFR gene was isolated from the F. dibotrys using the RACE (rapid amplification of cDNA ends) methods to scanning cDNA library; and bio-informatical analysis, and Southern blot were also made. 【Result】One DFR cDNA clones (FdDFR1) was cloned (GenBank accession No. EF522145/ EF522146). The comparison between cDNA and genomic DNA sequences revealed that FdDFR1 is composed of two exons and one intron. Southern blot analysis indicated that DFR belongs to a small gene family, and only one FdDFR1 copy in F. dibotrys genomes. A NADP binding site (VTGASGFVGSWLVMRLLEHGY) and a substrate specificity motif (TVNVEEKQKPVYDETCW SDVDFCR RV) were observed in the deduced amino acid sequence of FdDFR1 contained N-terminal region. 【Conclusion】It is concluded that FdDFR1 has the typical characteristics with other homologous genes.