穗醋栗MYB10基因克隆结构分析及功能验证

Cloning, structure analysis and functional verification of MYB10 in Ribes L.

摘要:

为探究花色苷合成相关转录因子MYB10在不同颜色穗醋栗果实着色差异的分子机理，通过cDNA末端快速扩增技术(rapid amplification of cDNA ends, RACE) 法从果实花青素含量有较大差异的黑穗醋栗(Ribes nigrum L.)、红穗醋栗(Ribes rubrum L.) 和白穗醋栗(Ribes album L.) 中分别克隆出MYB10基因，分别命名为RnMYB10 (KY786107)、RrMYB10 (KY786108)和RaMYB10 (MW660848)。系统发育分析表明，RnMYB10和RrMYB10在进化上具有同源性。实时荧光定量PCR (real-time quantitative PCR, RT-qPCR) 结果表明：黑穗醋栗各时期果实中MYB10表达量均高于红穗醋栗且远远高于白穗醋栗。随着果实直径加大颜色加深，RnMYB10和RrMYB10表达量呈现先上升后下降的趋势(在果实转色程度75%时达到最大值)，RaMYB10表达量极低，几乎无表达。过表达RnMYB10和RrMYB10的拟南芥呈现紫色叶柄和叶片，过表达RaMYB10的拟南芥无明显变化。说明MYB10基因在穗醋栗果实呈色中发挥重要作用。

Abstract:

This study aims to investigate the molecular mechanism of the transcription factor MYB10, which is involved in anthocyanin biosynthesis, in different colors of Ribes L. fruitification. Rapid amplification of cDNA ends (RACE) was used to clone the MYB10 genes from Ribes nigrum L. (RnMYB10), Ribes rubrum L. (RrMYB10), and Ribes album L. (RaMYB10), respectively. Phylogenetic analysis showed that RnMYB10 and RrMYB10 were evolutionarily homologous. Real-time quantitative PCR (RT-qPCR) showed that the expression of MYB10 in the fruits of Ribes nigrum L. was higher than that of Ribes rubrum L. and much higher than that of Ribes album L. The expression of RnMYB10 and RrMYB10 increased at first and then decreased as the fruit diameter increased and the fruit color deepened (the maximum expression level was reached at 75% of the fruit color change), while the expression level of RaMYB10 was very low. Overexpression of RnMYB10 and RrMYB10 in Arabidopsis thaliana resulted in purple petioles and leaves, whereas overexpression of RaMYB10 resulted in no significant color changes. This indicates that MYB10 gene plays an important role in the coloration of Ribes L. fruit.