红穗和白穗醋栗F3H的克隆及其在果实成熟过程中的表达分析

以红穗醋栗（Ribes rubrum L.）和白穗醋栗（R. albrum L.）果实为试材，采用RACE方法克隆黄烷酮3–羟化酶（F3H）基因cDNA全长序列，分别命名为RrF3H和RaF3H（KU984435和KU984436）。RrF3H全长1 351 bp，开放阅读框长1 098 bp，编码365个氨基酸；RaF3H全长1 291 bp，开放阅读框长1 098 bp，编码365个氨基酸。氨基酸多序列比对表明该基因编码的蛋白具有非血红素双加氧酶结构域（DIOX-N superfamily）和典型的F3H蛋白功能结构域（2OG-FeⅡ_Oxy加氧酶结构域），属于双加氧酶超家族。系统发育分析表明，RrF3H和RaF3H在进化上具有明显种属特性，属于相对独立的进化分支。定量PCR分析表明，F3H在红穗醋栗中表达量远远高于白穗醋栗，在红穗醋栗中随着果实着色加深表达量逐渐上升，果实着色约75%时表达量最高，之后下降；白穗醋栗中随着果实生长，该基因的表达下降，花色苷含量也呈下降趋势，说明F3H基因在醋栗果实着色过程中发挥作用。

Molecular Cloning and Expression Analysis of F3H Gene in Ribes rubrum and R.albrum During Fruit Mature

Red currant（Ribes rubrum L.）and white currant（R. albrum L.）fruits were used as materials，two full-length cDNA sequences of F3H gene were cloned using RACE（rapid amplification of cDNA ends），and denoted as RrF3H and RaF3H. Both of them contained a 1 098 bp open reading frame that encoded a deduced protein with 365 amino acid residues. Amino acid sequence alignment showed that the protein had a non-heme dioxygenase domain（DIOX-N superfamily）and 2OG-FeⅡ-dependent dioxygenase superfamily. Phylogenetic analysis showed that，RrF3H and RaF3H had obvious characteristics of species in evolution，which had an relative independent evolutionary branch. Quantitative RT-PCR analysis showed that，during the period of fruit development F3H expression in red currant was much higher than that in white currant. With the deepening of fruit colour，the expression level gradually increased until the period of 75% fruit colour，then the RrF3H decreased rapidly. With the fruit growth of white current，the expression pattern of F3H decreased. And the content of anthocyanins in the same period also showed a decreasing trend. All the results suggested that F3H play a role in the process of fruit coloring.