红花玉兰MYB家族基因MawuCPC的克隆及在花色调控中的作用分析

Cloning of MYB Family Gene MawuCPC of Magnolia wufengensis and Analysis of Its Role in Flower Color Regulation

红花玉兰是我国特有的珍稀园林观赏树种,其花色变异类型极为丰富,有深红、粉红和白色等一系列的花色变异品种,然而其花色调控的分子机制目前仍不清楚。为探究红花玉兰花色调控的分子机制,以不同色系的红花玉兰为试验材料,克隆获得了红花玉兰的花色调控关键候选基因MawuCPC,对其开展了系统进化分析、氨基酸序列结构分析、亚细胞定位试验、酵母双杂交试验、荧光素酶试验,并通过实时定量PCR比较分析了不同花色品种间MawuCPC的表达水平。结果表明,红花玉兰的MawuCPC基因编码区长度为258 bp,共编码85个氨基酸;系统进化分析显示,MawuCPC与拟南芥以及其他物种的CPC类成员共聚于同一进化分支,进一步支持了MawuCPC属于红花玉兰的CPC家族成员;序列结构分析进一步显示,MawuCPC蛋白仅具有1个保守的R3结构域和1个bHLH结合基序。亚细胞定位试验结果表明,MawuCPC蛋白特异性地定位于细胞核内。酵母双杂交和荧光素酶试验显示,MawuCPC和MawuPAP1均可以与红花玉兰的bHLH家族蛋白MawubHLH1发生相互作用,且MawuCPC与MawubHLH1的结合能够严重抑制MawuPAP1和MawubHLH1对结构基因MawuDFR启动子的表达增强作用。实时定量PCR结果显示,MawuCPC在红花玉兰白花系品种JB中表达水平最高,在粉花系品种娇艳中表达次之,在红花系品种娇红1号中表达最低。以上结果表明,在红花玉兰中,MawuCPC可能同样是通过与PAP1/2类蛋白竞争结合bHLH蛋白来发挥其花色调控的作用,且可能正是由于该基因的表达水平差异促使红花玉兰产生了极其丰富的花色变异类型。

Magnolia wufengensis is a rare ornamental tree species endemic to China.Its flower color variation types are extremely rich.There are a series of flower color variation varieties such as deep red,pink and white.However,the molecular mechanism of flower color regulation is still unclear.In order to explore the molecular mechanism of flower color regulation of Magnolia wufengensis,MawuCPC,a key candidate gene for flower color regulation of Magnolia wufengensis,was cloned from Magnolia wufengensis.Phylogenetic analysis,amino acid sequence structure analysis,subcellular localization experiment,yeast two-hybrid experiment and luciferase experiment were carried out.The expression level of MawuCPC in different flower color varieties was compared and analyzed by Real-time Quantitative PCR.The results showed that the coding region of MawuCPC gene was 258 bp,encoding 85 amino acids.Phylogenetic analysis showed that MawuCPC was clustered with CPC members of Arabidopsis thaliana and other species in the same evolutionary branch,which further supported that MawuCPC belonged to the CPC family of Magnolia wufengensis.Sequence structure analysis further showed that MawuCPC protein had only a conserved R3 domain and a bHLH binding motif.The result of subcellular localization experiment showed that MawuCPC protein was specifically localized in the nucleus.Yeast two-hybrid and luciferase assays showed that MawuCPC and MawuPAP1 could interact with the bHLH family protein MawubHLH1 of Magnolia wufengensis,and the binding of MawuCPC to MawubHLH1 could seriously inhibit the enhancement expression of MawuPAP1 and MawubHLH1 on the promoter of the structural gene MawuDFR gene.Real-time Quantitative PCR results showed that the expression level of MawuCPC was the highest in the white flower variety JB,followed by the pink flower variety Jiaoyan,and the lowest in the red flower variety Jiaohong 1.The above experimental results indicate that in Magnolia wufengensis,MawuCPC may also play its role in flower color regulation by competing with PAP1/2 protein to bind bHLH protein,and maybe the difference in the expression level of this gene promotes Magnolia wufengensis to produce extremely rich flower color variation types.