红肉苹果分裂素响应基因MdMYB308的克隆与表达分析

Molecular Cloning and Expression Analysis of Cytokinins Responsive Gene MdMYB308 in Red Flesh Apple

【目的】细胞分裂素是调控植物花青苷合成的重要激素,从新疆红肉苹果杂种一代优系‘紫红3号’中克隆得到分裂素响应基因Md MYB308,研究其在细胞分裂素调控苹果花青苷合成中的作用,为进一步完善红肉苹果育种的理论与技术体系提供参考。【方法】以红肉苹果‘紫红3号’(新疆红肉苹果与‘富士’杂交1代)的红色幼嫩叶片为外植体诱导的红色愈伤组织为试材,设计引物利用PCR克隆Md MYB308,对其进行生物信息学分析;并用不同浓度细胞分裂素处理,采用荧光定量PCR分析Md MYB308及花青苷合成相关基因的表达;通过酵母双杂交试验、荧光双分子互补试验验证Md MYB308与Mdb HLH3的互作关系。【结果】在‘紫红3号’中克隆获得Md MYB308全长,其包含768 bp完整的开放阅读框,编码255个氨基酸,预测其编码蛋白质分子量为28.37 kD,等电点为8.94;系统进化树分析表明,Md MYB308与At MYB4、Fa MYB1、At MYBL2在同一个进化枝上,氨基酸序列比对发现,Md MYB308蛋白存在EAR抑制序列;提高6-BA浓度有利于苹果愈伤组织花青苷的累积,与无细胞分裂素处理相比,1 mg·L^(-1) 6-BA处理愈伤花青苷合成结构基因Md CHS、Md DFR、Md UFGT与转录基因Md MYB10、Mdb HLH3的表达量升高,而Md MYB308表达被抑制;酵母双杂交与荧光双分子互补试验表明,Md MYB308与Mdb HLH3能相互作用。【结论】细胞分裂素(6-BA)可能通过抑制Md MYB308的表达影响Md MYB308与Mdb HLH3的结合从而促进花青苷的累积。

【Objective】Cytokinin is an important hormone in the regulation of anthocyanin synthesis in plants.To develop the theory and technology for red flesh apple breeding,the function in the cytokinin regulating anthocyanin metabolism of MYB transcription factor gene MdMYB308 in ＇Zihong No.3＇ from Malus sieversii f.neidzwetzkyana F1 population was studied.【Method】The callus induced from the leaves of ＇Zihong No.3＇ apple was used as materials.The MdMYB308 in ＇Zihong No.3＇ was cloned and its bioinformation was analyzed.The expression level of MdMYB308 and anthocyanin biosynthesis related genes in callus which grown on different concentrations of 6-BA was studied by the q RT-PCR.Meanwhile,the interaction between Mdb HLH3 and MdMYB308 was verified by yeast two-hybrid system and bimolecular fluorescence complementation assay.【Result】The full length of MdMYB308 in ＇Zihong No.3＇ was cloned,and the gene was 768 bp which encoded 255 amino acids.It was predicted that the molecular mass of this protein was 28.37 kD,and p I was 8.94.A phylogenetic tree indicated that MdMYB308,At MYBL2,Fa MYB1 and At MYB4 are located in the same evolutionary branch.The aligned protein sequences revealed that MdMYB308 contain the EAR motif.Furthermore,the content of anthocyanin rose as 6-BA concentration increased as well.The transcript levels of anthocyanin structural genes（Md CHS,Md DFR,Md UFGT） and transcription factors（Md MYB10,Mdb HLH3） were significantly higher in callus grown on 1 mg·L^（-1） 6-BA compared with 6-BA-deprived callus.In contrast,the expression of MdMYB308 was inhibited.The results of yeast two hybrid experiments and bimolecular fluorescence complementation assays showed that the MdMYB308 could interact with Mdb HLH3.【Conclusion 】Cytokinin（6-BA） could promote anthocyanin accumulation by down-regulating the expression of MdMYB308 which may destroy the combination of MdMYB308 and Mdb HLH3.