苹果属观赏海棠McDFR的克隆及不同叶色品种间的表达差异

Cloning and Expression of McDFR gene in the Different Foliar Color Cultivars of Malus crabapple

以苹果属观赏海棠品种‘王族’叶片总RNA为模板,通过RT-PCR与RACE扩增,获得1个1247bp的二氢黄酮醇-4-还原酶基因的cDNA序列,其基因编码区共1021bp,存在3个ORF,编码348个氨基酸,命名为McDFR。基因序列全长2396bp,有5个内含子,所有内含子的剪切位点均符合真核生物"GT-AG"规则。使用实时荧光定量PCR和紫外可见光分光光度计,对4种不同叶色类型的观赏海棠品种‘火焰’(绿色类叶片)、‘绚丽’(新叶有色类红色叶片)、‘高原之火’(新叶有色类红色叶片)和‘王族’(常色类紫色叶片)幼叶和功能叶中的McDFR表达及其花色苷和类黄酮的含量进行测定分析,结果表明:McDFR在4个品种的幼叶和功能叶中均有表达,McDFR表达量与花色苷积累有一定的相关性,McDFR在幼叶中表达量高于功能叶;在4个品种中,McDFR在‘高原之火’幼叶中表达量最大。幼叶中的类黄酮含量高于功能叶,McDFR表达量也大于功能叶。在功能叶中,除绿色叶品种‘火焰’外,类黄酮含量越高,McDFR的表达量越大,说明McDFR对苹果属观赏海棠叶片花色苷、类黄酮代谢途径及色泽形成具有重要作用。

Dihydroflavonol-4-reductase（DFR）is the first key enzyme in the later procedure of anthocyanins biosynthesis.With the total RNA drived from leaves of Malus crabapple＇Royalty＇as the template,the full cDNA of DFR gene（1 247 bp）was cloned by reverse transcription polymerase chain reaction（RT-PCR）and rapid-amplification of cDNA ends（RACE）.The gene is named as McDFR,containing three open reading frames（ORF,1 021 bp）,and encoding a protein of 348 amino acids.DNA sequence has 2 396 bp,containing five introns,and all the cleave sites obey with the GT-AG rule.The expression of McDFR and the content of anthocyanin and flavonoids were determined respectively by real-time quantitative PCR and spectrophotometer in the mature and young leaves of M.＇Flame＇（green young and mature leaf）,M.＇Radiant＇（red young leaf and green mature leaf）,M.＇Prairifire＇（red young leaf and green mature leaf）and M.＇Royalty＇（purple young and mature leaf）.The results showed that McDFR was expressed in both mature and young leaves of the above four cultivars.The trends of anthocyanin content were consistent with that of the relative expression of McDFR gene.The expression levels of McDFR in the young leaves were higher than that in the mature leaves.Among the four cultivars,the M.＇Prairifire＇in the young leaves showed the highest expression levels of McDFR.Flavonoid content in young leaves was higher than that in the mature leaves,and the expression of McDFR was also higher than in the mature leaves.In the mature leaves,except for the green leaf variety M.＇Flame＇,the higher content the flavonoid the higher expression level McDFR.These results have determined that McDFR plays an important role in anthocyanin and flavonoid metabolic pathways and the color formation in the leaves.