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二氢黄酮醇4–还原酶(dihydroflavonol 4-reductase,DFR)是花青素生物合成晚期阶段的关键酶,属于NAD/NADP依赖型还原酶家族,催化从二氢黄酮醇转变成无色花色素苷的反应。利用UV-A处理‘津田’芜菁(‘Tsuda’turnip)和‘赤丸’芜菁(‘Yurugi Akamaru’turnip)块根24 h后提取总RNA,通过RT-PCR方法分别克隆了‘津田’芜菁BrDFR1和‘赤丸’芜菁BrDFR2基因。BrDFR1和BrDFR2的开放读码框分别为1 158 bp和999 bp,分别编码385和332个氨基酸。氨基酸序列分析显示,BrDFR1和BrDFR2与大白菜DFR具有高度同源性,从第8到第302位氨基酸的肽段具有FR_SDR_e结构域。BrDFR1和BrDFR2基因组全长序列均含有5个位置与序列完全相同的内含子。Southern杂交结果显示,‘津田’芜菁BrDFR1和‘赤丸’芜菁BrDFR2基因均为单一拷贝。UV-A可以诱导BrDFR1基因表达,对BrDFR2基因表达的诱导效果不明显。BrDFR1和BrDFR2基因在大肠杆菌细胞中可以表达并纯化出分子量约为42.8 kD的BrDFR1蛋白和37.5 kD的BrDFR2蛋白。过量表达BrDFR1和BrDFR2基因的烟草花色加深。芜菁DFR基因的RNAi载体遗传转化烟草后,转基因植株的花色变浅。这些工作将为阐明依光型和非依光型花青素生物合成机理奠定研究基础。
Dihydroflavonol 4-reductase (DFR), a key enzyme in the late stage of anthocyanin biosynthesis, belongs to the NAD / NADP-dependent reductase family and catalyzes the conversion from dihydroflavonol to colorless anthocyanidins Glycoside reaction. Total RNA was extracted from roots of ’Tsuda’turnip’ and ’Yurugi Akamaru’turnip’ by UV-A treatment for 24 h, and RT-PCR methods were used to clone ’Tsuda’ turnip BrDFR1 and ’red pill’ turnip BrDFR2 genes. The open reading frames of BrDFR1 and BrDFR2 were 1 158 bp and 999 bp, encoding 385 and 332 amino acids, respectively. Amino acid sequence analysis showed that BrDFR1 and BrDFR2 have high homology with Chinese cabbage DFR, and peptides from amino acid 8 to 302 have FR_SDR_e domain. The full-length sequences of the BrDFR1 and BrDFR2 genomes contain 5 introns with exactly the same positions and sequences. Southern hybridization results showed that both ’Tsuda’ turnip BrDFR1 and ’red pill’ turnip BrDFR2 genes were single copy. UV-A can induce BrDFR1 gene expression, the effect of BrDFR2 gene expression was not obvious. The BrDFR1 and BrDFR2 genes can express and purify BrDFR1 protein with a molecular weight of about 42.8 kD and BrDFR2 protein with 37.5 kD in E. coli cells. Tobacco flowers overexpressing BrDFR1 and BrDFR2 genes deepened. Turnip DFR gene RNAi vector genetic transformation of tobacco, transgenic plants lighter. These work will lay the foundation for elucidating the mechanism of anthocyanin biosynthesis by light and non-light.