黑松抗松萎蔫病相关miRNAs及其靶基因的鉴定与功能研究

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the causative factor of pine devastating disease - pine wilt disease, and also an important invasive species and quarantine object. Currently, it has been mainly concentrated on the pathogen of PWN, the interaction between PWN and associated bacteria, pathogenic mechanism, the invasion mechanism of PWN and so on. Meanwhile some progress has also been made in the prevention and control of PWN disease and the breeding of traditional disease-resistant tree species. However, there are few studies on the disease resistance of host plants, especially the molecular disease resistance mechanism of host pine. This project intends to use Pinus thunbergii, an important afforestation and landscape tree species, as the research object. The sterile PWN-resistant Pinus thunbergii seedlings infected by Bursaphelenchus xylophilus are used as materials, to discover and identify miRNAs and their target genes related to PWN resistance, and carry on the functional analysis of miRNAs. Our aim is to obtain discrepant miRNAs and mRNAs in NBS-LRR domain of Pinus thunbergii seedlings infected by Bursaphelenchus xylophilus, accompanied by molecular biological identification and functional verification, and further study on the intermodulation pattern of miRNA482 family and NBS-LRR-R genes, in order to explore the regulation mechanisms of the miRNAs involved in disease resistance. This study will lay the foundation for improving Pinus thunbergii resistance to Bursaphelenchus xylophilus stress and cultivating disease-resistant seedlings by molecular biology methods.

松材线虫是松树毁灭性病害-松萎蔫病的致病因子，也是一重要的入侵物种和检疫性对象，目前研究主要集中在松材线虫、线虫与伴生细菌互作及致病机制、松材线虫入侵机制等方面，同时对松材线虫病的防治研究和传统抗病树种选育方面也取得一些进展，但是对于其寄主植物的抗病反应研究较少，尤其是寄主松树的分子抗病机理。本项目拟以黑松这一重要的造林和景观树种为研究对象，以松材线虫侵染的黑松抗性品系无菌幼苗为材料，通过测序与实验检测方法，发掘和鉴定调控黑松抗松材线虫相关的miRNA及其靶基因，并进行miRNA功能分析；获得松材线虫侵染胁迫下，黑松NBS-LRR结构域差异mRNA和miRNA，并进行分子生物学鉴定和功能验证；研究miRNA482家族与NBS-LRR类R基因的互调模式，探索黑松幼苗miRNA参与抗病过程的调控作用机制。研究结果将为利用分子生物学手段提高黑松抗松萎蔫病能力及培育抗病苗木奠定基础。

松材线虫是松树毁灭性病害-松萎蔫病的致病因子，对其寄主植物的抗病反应研究，尤其是寄主松树的分子抗病机理研究具有重要的意义。本项目分离培养了野生松材线虫，研究了松材线虫的致病性，并探索了黑松幼苗的培养和侵染方法。构建了黑松幼苗转录组文库、Small RNA文库和降解组文库，完成测序和生物信息学分析。获得了768个差异表达基因（336上调，432下调）。筛选到显著差异表达的抗病相关NBS-LRR类基因13个。鉴定出789个miRNAs，其中包括406个已知miRNAs和383个新novel-miRNAs，显著差异miRNA有42条（26上调，16下调），并进行了靶基因预测。筛选出部分抗病相关miRNA&靶基因，这些靶基因编码转录因子或抗病蛋白。转录组与miRNA联合分析筛选出12个miRNA-mRNA对。其中novel-m0263-5p和novel-m0317-5p可能与黑松的防御系统有关联，其预测靶基因unigene0016763和unigene0041830的功能注释都为LRK1。qRT-PCR结果表明，松材线虫侵染前后，黑松幼苗中novel-m0317-5p与unigene0041830的表达量始终呈负相关。克隆了unigene0041830基因和novel-m0317-5p片段。novel-m0317-5p瞬时转染黑松幼苗，接种松材线虫后表现出明显的松萎蔫病症状。结果表明，novel-m0317-5p与黑松幼苗的抗病性成负相关，novel-m0317-5p过表达导致黑松抗病性明显下降。研究结果为利用分子生物学手段提高黑松抗松萎蔫病能力及培育抗病苗木奠定了基础。