马铃薯诱导表达型基因StuPPO9及相关miRNA在抗晚疫病中的作用分析

The researches about Polyphenol oxidase (PPO) focus on the enzymatic browning reaction, while the known of other physiological functions is rare. Our pre-test showed StuPPO9 is the only gene that highly induced in response to pathogen infection, especially to Phytophthora infestans. The signal molecules, MeJA and SA, have a positive regulation on it. Meanwhile, the potential miRNAs regulating StuPPO9 are mostly related to disease resistance. Therefore, we predict StuPPO9 may be involved in Phytophthora infestans-defense. However, its roles are not clear. In this proposal, we will research on the differences of disease degree and other physiological indicators between transgenic and wild-type potato after Phytophthora infestans infection by stablely over-expressed StuPPO9. Furthermore, the resistant genes related StuPPO9 and the miRNAs regulating StuPPO9 will be identified by transcriptome and small RNA sequencing followed by miRNAs-mRNAs integration analysis and other strategies. The results will be verified by transient expression and RT-qPCR techniques. This work will reveal the function of StuPPO9 and its related miRNAs in Phytophthora infestans-defense. It may provide a theoretical basis for using key nodes of miRNAs mediation to obtain continuously disease-resistant varieties.

多酚氧化酶的研究侧重于褐变反应，而对其它生理功能的研究相对空白。申请人发现，马铃薯PPO家族中StuPPO9是唯一受晚疫病原菌诱导大量表达的基因，抗病信号分子MeJA/SA对其有正向调控作用。同时潜在调控StuPPO9的miRNAs也多与抗病性相关。故推测StuPPO9可能参与了植物抗晚疫病的过程，但作用机制尚不明确。本项目通过在马铃薯中稳定过表达StuPPO9，测定病原菌侵染后转基因和野生型植株的病害程度及抗性生理等指标，验证StuPPO9在抗晚疫病中的作用。通过转录组和小RNA测序，结合miRNAs-mRNAs整合分析等策略，利用瞬时表达和RT-qPCR等技术，鉴定并验证StuPPO9与抗性相关基因的联系、确定调控StuPPO9的miRNA。该研究将揭示StuPPO9参与植物抗性的功能以及miRNA对其调控作用，为后续利用关键节点miRNA介导获得持续抗病品种提供理论支持。

多酚氧化酶的研究侧重于褐变反应，而对其它生理功能的研究相对空白。项目前期通过检索更新的马铃薯基因库，发现马铃薯多酚氧化酶基因家族包含10个成员。其中StuPPO9是唯一受晚疫病原菌诱导大量表达的基因。本项目实施中，通过启动子诱导试验和基因表达分析试验，确定了StuPPO9基因为诱导表达型基因，其受晚疫病诱导表达。通过构建StuPPO9基因过表达载体与CRISPR/Cas9基因编辑载体，利用农杆菌介导法获得烟草及马铃薯转基因阳性植株，通过对比野生型和转基因型染病后情况，证明了StuPPO9基因具有抗病作用。为探讨StuPPO9的抗病机理，利用转录组学测序技术，从晚疫病原菌侵染的转基因植株与野生型植株中，筛选出了差异表达基因与共表达基因，并利用qRT-PCR技术验证了这一结果。同时，通过psRNATarget靶基因预测和miRanda结合分析，结合GO、KEGG数据库，寻找出了抗病途径中潜在调控StuPPO9基因的miRNAs，并采用qRT-PCR技术对相关miRNA进行了表达分析。该项目获得的结果，可为后续进一步研究马铃薯抗病机制，以及马铃薯抗晚疫病育种提供理论依据。