新疆红肉苹果杂种一代红肉株系类黄酮和着色差异机理的研究

选育类黄酮含量高、可鲜食或加工的功能型苹果是发展方向。本申请在已构建了新疆红肉苹果F1和BC1分离群体、明确了实生株系MdMYB10启动子基因型与表型特征以及通过RNA-seq等获得MdMYB4/12/22/16/111等5个候选正负调控转录因子的基础上，进一步以果肉颜色及类黄酮含量存在明显差异的新疆红肉苹果杂种一代４个红肉株系为试材，通过基因克隆和生物信息学分析等研究候选基因结构特征和时空表达特性及其与4个红肉株系果实类黄酮含量变化的关系；利用酵母双杂交等技术在离体和活体状态下研究候选基因与bHLH3等转录因子的蛋白互作，利用EMSA等技术在离体和活体条件下探讨候选基因与CHS等类黄酮生物合成结构基因启动子在蛋白-DNA水平上的互作特点及调控作用，并进行转基因功能验证及GFP亚细胞定位，从而探讨苹果红肉株系果实类黄酮与着色差异的分子机理，为完善功能型苹果育种的理论与技术体系提供科学依据。

Breeding functional apple with high flavonoid content for fresh and processing is the development direction of apple breeding. On the basis of building F1 and BC1 segregation population of Xinjiang wild red-flesh apple [Malus sieversii f. neidzwetzkyana（Dieck）Langenf.], identification the relationship between phenotypic traits of hybrids and MYB10 promoter genetypes, and obtaining five MYB transcription factors, MdMYB4, MdMYB12, MdMYB16, MdMYB22 and MdMYB111, which might positively or negatively regulate flavonoid synthesis by RNA-seq, we will clone and perform bioinformatics analysis of these five candidate transcription factors and study their spatial and temporal expression characteristics in two R6R1 genetype strains named 'Hongcui 1' and 'Hongcui 4' with low flavonoid content in fruit flesh and two R6R6 genetype strains named 'Zihong 1' and 'Zihong 2' with high flavonoid content in fruit flesh. Further, yeast two hybrid technology and other technology will be used to study the protein-protein interaction between these five candidate transcription factors and other flavonoid synthesis regulation transcription factors, such as bHLH3, in vitro and living conditions; EMSA and ChIP-PCR technique will be used to study the protein-DNA interaction characters and regulation functions between five candidate transcription factors and structure genes promoter of flavonoid synthesis, such as CHS, in vitro and living conditions. Their function in plant will be verified in transgenic plants. These results will elucidate the biosynthesis and regulation mechanism of flavonoid in hybrid progeny fruits of Malus sieversii f.neidzwetzkyana, and lay the theoretical basis of functional apple breeding.