苹果受体类蛋白激酶MdCERK1与MdPR4互作调控砧木再植病抗性的分子机制研究

开展苹果砧木再植病抗性基因筛选及信号路径解析，对提高再植病防治的准确性、缩短育种周期均具有重要意义。项目组前期研究成果表明，苹果的MdCERK1和MdPR4基因能响应再植病病原菌侵染，且两者在该植病体系中存在互作关系。在此基础上，本项目拟通过Y2H技术对两者的互作结构域进行鉴定；利用LUC/REN技术验证几丁质对两者互作强度的影响；通过几丁质亲和性实验鉴定MdPR4的几丁质绑定能力；通过对MdCERK1沉默和过表达的转基因苹果接种再植病主要致病真菌后，观察表型和检测下游基因表达水平，明确MdCERK1的抗病功能；通过对共转MdCERK1（-）× MdPR4（+）的苹果愈伤进行几丁质处理，检测下游基因表达量，明确两者对几丁质信号的转导机制。综合上述多方面的证据，解析苹果MdCERK1通过与MdPR4互作调控再植病抗性的分子机理，获得的转基因砧木品种，也将为防治方法开发和抗性品种培育奠定基础。

It is of great importance for enhancing the precision of replanting control and shortening the breeding cycle to carry out research on apple rootstocks about ARD-resistant gene screening and signal pathway clarification. Our previous results have shown that MdCERK1 and MdPR4 can response to ARD pathogen inoculation and interact with each other. On this basis, Y2H will be used to identify the exact domains involved in the interaction. Meanwhile, how chitin affects the interaction intensity will be examined by LUC/REN. Additionally, MdPR4’s chitin binding ability will be verified by chitin affinity assay. To clarify MdCERK1’s function in ARD resistance, phenotypes and downstream genes’ expression levels will be compared after inoculating MdCERK1’s knock out and overexpression transgenic lines with ARD major fungal pathogens. Finally, through chitin treatment on MdCERK1 (-) × MdPR4 (+) bi-transgenic apple callus, downstream genes’ expression levels will be tested to define their transduction mechanism for chitin signal. Collectively, these findings will reveal the molecular mechanism of how MdCERK1 and MdPR4 interact with each other to regulate ARD resistance. Moreover, the transgenic rootstocks obtained from this project will lay a foundation for control methods development and resistant varieties breeding.