MdRAf5基因介导的苹果砧木干旱抗性分子调控网络解析

China is the biggest apple production country in the world，but the dominant producing areas of apple are mostly distributed in arid and semi-arid areas. So it is extremely important to study drought resistant rootstocks and their resistance molecular mechanism. A gene (MdRAf5) isolated from multi-resistant apple rootstock ‘Qingzhen 1’ by this research team was found to be related to anti-stress resistences. Up-regulation of MdRAf5 in Arabidopsis thaliana transgenic plants significantly increased the drought resistance. In this project, biological functions of MdRAf5 are studied to elucidate drought resistance by transgenic means. Furthermore, RNA-seq approach will be used as gene expression profiles to reveal the mechanism of drought resistance induced by MdRAf5 using transgenic apple plants of overexpressing and RNAi. Different techniques including the yeast two-hybrid technology, bimolecular fluorescence complementation and in-gel kinase assay will be used to examine the interaction-proteins of MdRAf5 and the downstream targets in the signal pathway of drought resistance. By analyzing the components of MdRAf5 involved signaling pathways, the molecular network of related to drought resistance in apple rootstock are established. This study would not only clarify the molecular mechanism of MdRAf5 involved in regulating drought resistance of apple rootstock, but also lay theoretical foundation for the genetic engineering breeding of apple rootstock.

我国是世界上最大的苹果生产国，但我国苹果优势产区多分布在干旱和半干旱地区，因此耐旱性砧木品种的选育及其抗性机理研究对提高苹果产量并改善苹果品质既具有重要的理论意义也有潜在的推广价值。本团队从自主选育的无融合多抗性砧木‘青砧1号’中克隆到关键抗旱性基因-MdRAf5，前期研究表明超表达MdRAf5显著提高了拟南芥植株的干旱抗性。本研究拟利用分子生物学手段，深入研究MdRAf5基因的抗旱功能；通过转录组技术（RNA-seq）比较干旱胁迫下MdRAf5基因过量表达和沉默苹果株系的基因表达差异，揭示其干旱调控机理；利用酵母双杂交、双分子荧光互补等技术，分离鉴定MdRAf5的下游级联组分，建立苹果砧木MdRAf5介导的干旱胁迫级联应答信号通路，探讨通路中的成员抗旱功能。最后，全面解析苹果砧木干旱抗性的分子网络，为苹果砧木的抗性育种提供理论依据。

我国是世界上最大的苹果生产国，但我国苹果优势产区多分布在干旱和半干旱地区，因此耐旱性砧木品种的选育及其抗性机理研究对提高苹果产量并改善苹果品质既具有重要的理论意义也有潜在的推广价值。本团队从自主选育的无融合多抗性砧木‘青砧1号’中克隆到关键抗旱性基因-MdRAf5，前期研究表明超表达MdRAf5显著提高了拟南芥植株的干旱抗性。本研究利用分子生物学手段，深入研究MdRAf5基因的抗旱功能；通过转录组技术（RNA-seq）比较干旱胁迫下MdRAf5基因过量表达和沉默苹果株系的基因表达差异，揭示其干旱调控机理；利用酵母双杂交、双分子荧光互补等技术，筛选并验证了MdRAf5的互作蛋白，选到MdRAf5下游的MdMAPKK4和MdMAPK11，初步明确了MdRAf5-MdMAPKK4-MdMAPK11级联途径；磷酸化实验证明MdMAPKKK11与MdNF-YB17互作后，可将MdNF-YB17磷酸化； 同时，对MdNF-YB基因家族进行了深入的生物信息及生物学分析，深入解析了MdRAf5调控苹果砧木干旱抗性的分子机制。为苹果砧木的抗性育种提供理论依据。