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以‘嘎拉’苹果(Malus × domestica‘Royal Gala’)为研究材料,利用同源克隆和PCR技术,分离了苹果细胞分裂素响应因子(cytokinin response factor)基因MdCRF4。该基因开放阅读框(ORF)为1077 bp,编码含有358个氨基酸的蛋白。保守结构域和系统进化树分析显示,MdCRF4蛋白包含一个保守的CRF结构域和一个保守的AP2/ERF结构域,与梨PbCRF4同源性最高。基因表达分析显示,该基因主要在苹果根和叶中表达并且响应细胞分裂素。EMSA试验显示MdCRF4原核表达蛋白能够绑定DRE(ACCGAC)序列。在‘王林’苹果愈伤组织中超表达MdCRF4,其花青苷积累显著增加,表明MdCRF4在调控花青苷积累中发挥重要作用。
Using Malus × domestica’Royal Gala as the research material, we isolated the apple cytokinin response factor gene MdCRF4 by homology cloning and PCR. The open reading frame (ORF) of this gene is 1077 bp, which encodes a protein of 358 amino acids. Conserved domains and phylogenetic tree analysis showed that the MdCRF4 protein contains a conserved CRF domain and a conserved AP2 / ERF domain, and has the highest homology with pear PbCRF4. Gene expression analysis showed that the gene is mainly expressed in apple roots and leaves and responds to cytokinin. The EMSA assay showed that the prokaryotic expression of MdCRF4 binds to the DRE (ACCGAC) sequence. Over-expression of MdCRF4 in ’Wanglin’ apple callus resulted in a significant increase of anthocyanin accumulation, indicating that MdCRF4 plays an important role in the regulation of anthocyanin accumulation.