苹果MdHCT17.2优异等位基因发掘及其调控果实绿原酸的功能分析

Apple fruit containing a lot of chlorogenic acid, although it has great benefits for health, it is the substrate in fruit browning occurred. If the genetic factors of controling chlorogenic acid and the regulated molecular mechanism were depth researched, the effective measure of achieve a goal will be obtained. The goal is fresh apples contenting high chlorogenic acid for high health value, processing apples contenting low chlorogenic acid for low browning. Although the QTLs of chlorogenic acid were reported, and the MdHCT was may considered candidate gene, it is not found that the relationship of the gene and genetic mechanism of chlorogenic acid in apple .The applicant obtained that there are closely relationship of the mutations site of promoter and coding region in MdHCT17.2 (MDP0000371737) and the chlorogenic acid character in apple by QTL mapping and candidate gene screening and analysis. In order to find out the most important mutation sites for the phenotype, this study intends to association analysis based on the gene in different apple accessions, and the mutations were selected to be the most related to chlorogenic acid content. Using the point mutation technique, the function of the mutation site will be verified by transgenic. This way can compare the contribution of multiple mutations of the gene to the phenotype, and excavated excellent allele, understanding its function. The study not only further understanding the molecular mechanism of the content of apple chlorogenic acid, but also for the genetic improvement of apples of functional nutrition and processing, genetic resources and theoretical basis provided.

苹果果实中含大量的绿原酸，有良好的保健作用，也是褐变发生的主要底物。对控制其的遗传因子深入研究，解析其分子机制，既是实现鲜食苹果高保健价值，又是实现加工苹果低褐变的有效途径。目前苹果绿原酸已有QTL定位结果，认为MdHCT为候选基因，但未见该基因与苹果绿原酸遗传机制关系的研究。申请人通过QTL定位和候选基因筛选分析，得到MdHCT17.2（MDP0000371737）的启动子和编码区变异，与苹果果实绿原酸性状形成密切相关。为了挖掘出对表型最有贡献的变异位点，本研究拟用不同苹果种质开展基于该基因的关联分析，筛选出与绿原酸含量变化最关联的变异位点，利用点突变技术，作转基因验证变异位点的功能，从而可比较该基因多个突变位点对表型的贡献，挖掘出优异等位基因，并验证其功能。该研究不仅对苹果绿原酸差异形成的分子机理有更深的认识，也为功能营养型和加工型苹果的遗传改良，提供基因资源和理论依据。

苹果果实中含有大量的多酚，绿原酸是其中的重要成分，有良好的保健作用，亦是褐变发生的主要底物。对控制苹果多酚和绿原酸的遗传因子进行深入研究，解析其分子机制，是实现鲜食苹果高保健价值，加工苹果低褐变的有效途径。. 本项目测定了大量的苹果资源果实的多酚组成。表明绿原酸是苹果多酚的主要组成，在野外和地方存在极有潜力的苹果多酚优异种质资源。开展苹果资源多酚组分的抗氧化特性分析后，得到中国地方品种有丰富的绿原酸，有极高的保健价值待开发，同时对本重点关注的多酚绿原酸的高、低含量的种质资源进行深入研究，为挖掘其中的基因资源提供了有价值的材料线索。对苹果芽变资源进行转录分析，筛选到一个转录因子MdBPC6，该基因为ATP结合转运基因对苹果发育和环境相应有关，与苹果多酚绿原酸的性状形成、苹果发育密切相关。对苹果绿原酸候选基因MdHCT17.2（MDP0000371737）进行了定位验证，得到绿原酸主效QTL峰值与BSA-seq技术连锁分析的G17.5 -CA重合，在对高密图谱进行重新的图谱整理，证实了该QTL的真实性。发现了该基因的上游启动子序列突变，有可能该突变是绿原酸性状变化的主要原因之一。相关基因型的功能分析已基本完成，由于试材的特殊性，数据正在汇总整理中，初步分析后，结果验证启动子差异对基因功能的差异有明显的贡献。. 这些研究结果能对苹果绿原酸表型的形成有更深入的认识，对苹果绿原酸差异的分子机理有更深的了解，为开发利用我国的苹果资源提供了有价值的切入点，也为功能营养型苹果和加工型苹果的遗传改良，提供基因资源和理论依据。