红叶山茶品种花青素苷相关基因表达水平及代谢产物分析

Analysis of Gene Expressing and Metabolites for Synthesising Anthocyanins Influencing the Red Color of Leaves of Camellia Cultivar

[目的]通过对花青素苷相关合成基因的表达水平和代谢产物的分析,系统地阐明‘金华美女’叶色变异与基因表达的关系。[方法]以‘金华美女’为材料,‘贝拉大玫瑰’、杜鹃红山茶和红山茶为参照组,使用NCBI Primer Designing Tool设计山茶DFR、ANS、LAR、ANR和UFGT基因的荧光定量引物,使用实时荧光定量PCR仪测定这5个基因在叶片4个发育时期的表达量;采用高效液相色谱仪测定对应时期的多酚合成途径的主要次生代谢产物(儿茶素、表儿茶素),以及花青素苷合成途径的主要代谢产物矢车菊素-3-O-葡萄糖苷的含量。[结果]表明:(1)在4个时期中,红叶品种叶片中DFR基因表达量与对照组差异不显著,ANS、LAR、ANR和UFGT基因在4个时期的表达量与对照组存在显著差异,这说明‘金华美女’类黄酮代谢途径相关基因在表达水平上发生了改变;(2)‘金华美女’叶片中多酚含量明显低于对照组,其中,表儿茶素含量仅为0.04 0.21 mg·g-1,这说明在生理水平上,‘金华美女’叶片中多酚合成途径可能受阻;(3)‘金华美女’叶片中矢车菊素-3-O-葡萄糖苷含量达1.2 1.4 mg·g-1,4个生长阶段均显著高于对照组,这说明‘金华美女’叶片具备持续合成花青素苷的能力。[结论]根据试验结果,推断‘金华美女’叶色变异的主要原因可能是由于花青素还原酶基因的表达水平受到了抑制,降低了矢车菊素催化成表儿茶素的效率,使叶片类黄酮代谢途径中以多酚为主的合成方向转向花青素苷合成方向。

[Objective] By analyzing the quantitative change of structural genes and metabolite of anthocyanin in 4 growth stages, the relationship between leaf-color mutation and gene expression was systematically illustrated. [Method] The leaves of Camellia japonica ‘Jinhua Meinü’ were selected taking the leaves of C. japonica ‘Nuccio＇s Bella Rossa’, C. azalea, and C. japonica as control. NCBI Primer Designing Tool was used to design the primers and the gene expressing of DFR, ANS, UFGT, ANR and LAR were analyzed by real-time PCR and measured the contents of catechins, epicatechins and cyanidin 3-O-glucoside by HPLC. [Result] （1） In the four growth stages, no significant difference was found in the expressing of DFR, nevertheless, the expressing of ANS, UFGT,ANR and LAR between red and green cultivar were obviously different, which indicating the express levels of those genes of flavonoids metabolic pathways had been altered in the leaves of C. japonica ‘Jinhua Meinü’. （2） The content of polyphenols in the leaves of C. japonica ‘Jinhua Meinü’ were at a significantly lower level than the control groups, in particular, the content of epicatechins was only 0.04-0.21 mg·g^-1, which suggesting the synthetic route of polyphenols were astricted in physiological level. （3） The average content of cyanidin 3-O-glucoside in the leaves of C. japonica ‘Jinhua Meinü’ was 1.2-1.4 mg·g^-1, which was at a significantly higher level than the control groups in all the four growth stages. The content of cyanidin 3-O-glucoside could explain the capacity of synthesizing anthocyanins was kept in the leaves of the red cultivar. [Conclusion] According to above data, it can be preliminary inferred that the leaf color of C. japonica ‘Jinhua Meinü’ related to the change of anthocyanins metabolic pathway.