穗醋栗dfr的克隆及其在果实成熟过程中的表达

对穗醋栗花色苷合成的分子机理知之甚少。拟探究穗醋栗花色苷合成关键基因dfr对不同颜色醋栗花色苷的影响,以黑穗醋栗(Ribes nigrum L.)、红穗醋栗(Ribes rubrum L.)和白穗醋栗(Ribes albrum L.)果实为试材,通过RACE方法克隆二氢黄酮醇4-还原酶(dfr)基因cDNA全长序列,分别命名为Rndfr、Rrdfr和Radfr(KY786100、KY786101和KY786102)。系统发育分析表明,Rndfr、Rrdfr和Radfr在进化上具有较高的同源性。测定果实发育不同时期的花色苷含量,结果显示,黑穗醋栗和红穗醋栗花色苷含量较高且随着果实的发育成熟而逐渐增加。而白色醋栗中花色苷含量极低,几乎检测不到花色苷。定量PCR分析表明,dfr在黑穗醋栗中的表达量在果实成熟的各个时期均高于红穗和白穗醋栗。随着果实直径不断变大和果皮着色加深,在黑穗醋栗中,dfr的表达量总体呈现持续上升的趋势;在红穗醋栗中,果实着色约75%时dfr的表达量最高,之后下降;在白穗醋栗中,dfr的表达量总体呈现下降趋势,其表达量最低。推测dfr基因在醋栗果实呈色中发挥作用。

Cloning and expression of dfr in Ribes L. during fruit maturation

Little is known about the molecular mechanism of currant anthocyanin synthesis. We investigated the effect of dfr, a key gene for anthocyanin synthesis in currant, on anthocyanins of different color currant. Black currant (Ribes nigrum L.), red currant (Ribes rubrum L.) and white currant (Ribes albrum L.) were used as test materials to determine the anthocyanin content at different stages of fruit development. Three full-length cDNA sequences of dfr gene were cloned by RACE (Rapid amplification of cDNA ends), and named as Rndfr, Rrdfr and Radfr. Phylogenetic analysis shows that Rndfr, Rrdfr and Radfr had high homology in evolution. The determination of anthocyanin content in different stages of fruit development shows that the content of anthocyanin in black currant and red currant was higher and gradually increased with the ripening of the fruit. While the content of anthocyanin in white currant was extremely low, and almost no anthocyanin was detected. Quantitative RT-PCR analysis shows that the expression level of dfr in black currant was higher than red currant and white currant in each period of fruit development. As the diameter of the fruit increased and the color of the peel deepened, the expression level of dfr in the black currant showed an increasing trend. In the red currant, the expression level gradually increased until the period of 75% fruit color, then the Rrdfr decreased rapidly. In white currant, the overall trend showed a downward trend, and its expression level was the lowest. All the results suggest that dfr gene plays a role in the process of fruit color.