苹果锈果类病毒源小RNA介导的MdPIF1调控苹果花青苷合成机制研究

Apple scar skin disease characterized with dapple symptom, which is induced by Apple Scar Skin Viroid (ASSVd), is one of the most serious destructive diseases in apple cultivation and production. Anthocyanin biosynthesis and regulation play critical roles in dapple symptom development, however, the mechanism of how these symptoms are induced by ASSVd remains unclear. According to our preliminary data, the viroid-derived small RNA vsiR693 targets and degrades the bHLH transcription factor MdPIF1, and potential MdPIF1-binding sites are present in the promoter of MdHY5-like, which plays a critical role in regulating anthocyanin biosynthesis. Thus, we hypothesize that the viroid might induce the symptom via the following possible route: vsiR693→MdPIF1→MdHY5-like→anthocyanin biosynthetic genes→fruit coloration. To validate our hypothesis, we will take advantage of the methods related to molecular biology, genetics, physiology and biochemistry to perform the experiments. Firstly, we will test the cleavage of MdPIF1 by vsiR693, and we will next verify the transcriptional activation of MdHY5-like induced by MdPIF1 and also the transcriptional activation of anthocyanin biosynthetic genes induced by MdHY5-like. Finally, we will confirm the roles of MdPIF1 and MdHY5-like in regulating anthocyanin biosynthesis by analyzing the transgenic plants, and unmask the possible mechanism of ASSVd induced dapple symptom in Malus domestica. This project will provide theoretical basis for development of antiviral strategy in Malus domestica, and also indicate new angels in investigating anthocyanin biosynthesis regulation.

苹果锈果类病毒引起的花脸病是苹果生产中一种毁灭性病害。花青苷合成及调控在该症状形成过程中扮演重要角色，但类病毒影响花青苷合成并导致花脸症状产生的机理仍不清晰。前期研究显示类病毒源的vsiR693能靶向转录因子MdPIF1，而MdHY5-like启动子中含有MdPIF1可结合的元件。因此，类病毒可能通过vsiR693→MdPIF1→MdHY5-like→花青苷合成相关基因→果实着色的途径调控花青苷合成和果实着色。基于此，本项目拟采用分子生物学、遗传学和生理生化技术，首先验证vsiR693对MdPIF1的靶向切割，然后鉴定MdPIF1对MdHY5-like的转录调控以及后者对花青苷合成结构基因的转录调控，最后利用转基因材料验证MdPIF1和MdHY5-like参与小RNA介导的类病毒调控花青苷合成并诱导花脸症状产生的过程。该项目从新视角研究花青苷合成的调控途径，为苹果抗病毒研究奠定理论基础。

类病毒引起的花脸病是苹果生产中常见的一种病害。该病害主要表现为果皮呈圆斑状变绿、变黄等症状，直接影响苹果的外观品质，降低经济价值。花青苷合成及调控在该症状形成过程中扮演重要角色，但类病毒影响花青苷合成并导致花脸症状产生的机理仍不清晰。在本项目中我们利用前期研究中鉴定的类病毒源小RNA vsiR693能靶向bHLH转录因子MdPIF1及MdPIF1通过转录调控花青苷合成结构基因的通路探究类病毒影响苹果果皮花青苷合成的可能机制。本项目研究结果表明，表现花脸症状的果皮中花青苷含量显著低于健康果皮，且花青苷合成相关基因表达量显著降低。我通过小RNA测序鉴定到类病毒源小RNA vsiR693及其靶基因MdPIF1。利用5’-RLM RACE技术和人工小RNA瞬时转染烟草技术证实vsiR693能够靶向切割MdPIF1。而通过注射果皮试验发现，过表达MdPIF1促进果皮花青苷合成，而通过TRV系统沉默MdPIF1的表达则抑制果皮花青苷的合成。进一步的，我们利用酵母单杂交、ESMA试验证实MdPIF1能够结合花青苷合成结构基因MdF3H和MdPAL的启动子序列中的G-box（5’-CACGTG-3’）基序。而利用荧光素酶报告基因和GUS染色试验证实MdPIF1能够能够转录激活二者的表达。此外，在果皮瞬时注射人工小RNA vsiR693能够抑制果皮花青苷的积累及MdPIF1和花青苷合成相关基因的表达。以上研究结果表明类病毒源小RNA vsiR693能够通过靶向降解MdPIF1，抑制花青苷合成结构基因MdF3H和MdPAL的表达，进而抑制花青苷的合成。本项目在分子层面解析了类病毒通过MdPIF1调控花青苷合成的内在机制，为深入了解类病毒导致苹果花脸症状形成提供了一定的依据。