miRNA对青稞抗条纹病基因的调控机制研究

The leaf stripe disease of hulless barley (H. vulgare subsp．Vulgare) is caused by a fungal pathogen, Pyrenophora graminea. The disease occurs widely and severely affects the yield and quality. Plant miRNAs play an important role in the development of plant diseases by regulating the expression of genes involved in resistance or susceptibility. A better understanding of the miRNA role plays in the development of the leaf stripe disease of hulless barley may lead to novel approaches to control this disease. In our previous work, 185 new miRNAs have been found by sequencing and analyzing in hulless barley. Among the miRNAs identified, hba-miR-new1 shows high levels of homology to Rdg2a. So it is thought to be regulated Rdg2a. Also, a new miRNA (hba-miR-new2) has a predicted target in AGO1 and is upregulated significantly after infection by the leaf stripe fungus. AGO1 is an essential catalytic component for miRNA to down-regulate gene expression levels. We hypothesized that the up-regulation of hba-miR-new2 miRNA in hulless barley down regulates AGO1 expression, and ultimately promotes the expression levels of genes involved in resistance or susceptibility by reducing the level of other miRNA species. In this study, we plan to systemically discover miRNA species involved in the resistance or susceptibility of hulless barley to P. graminea, identify their gene targets, validate the regulation of the target genes by these miRNA, and investigate the roles of these miRNAs (including hba-miR-new1 and hba-miR-new2) in the disease development. The proposed research will generate sufficient data to identify candidate miRNA species related to the stripe disease resistance and provide guidance in the breeding for strip resistance in hulless barley.

条纹病是青稞种植中最广泛的一种病害，严重影响其产量和品质，但对其发病机理了解甚少。miRNA能够靶向调控抗病/感病基因，从miRNA调控病害发展入手研究植物发病和抗病机理是一个新的角度。通过高通量测序分析，在青稞中发现185个新miRNA。其中miRNA （hba-miR-new1）与Rdg2a基因存在同源序列，推测该miRNA可能靶向调控抗病基因Rdg2a；一个新的miRNA（hba-miR-new2）的表达量随条纹病感染程度的加重而显著升高，预测其靶基因为AGO1。AGO1蛋白已被证明在植物抗病中发挥重要作用，是miRNA调控靶基因过程中的关键因子。我们将系统挖掘青稞抗条纹病相关的miRNAs，验证他们的功能，探究这些miRNAs及其调控的靶基因参与青稞条纹病的发病和抗病机理，有利于深入了解miRNA在植物病害发展中的作用，也有利于条纹病的防控和抗病新品种的选育。

青稞是藏族同胞的主粮，在青藏高原被广泛种植，具有丰富的营养价值和医药保健作用。条纹病是严重影响青稞产量与品质的最主要的病害之一，但对其抗病调控机理了解甚少。本研究以抗病品种的“昆仑14号”和敏感型品种“Z1141”为材料，首次对其条纹病感染前后的小RNA和mRAN进行了测序。共鉴定出24个差异表达miRANs （包括11个保守的miRNAs和13个新的miRNAs），24个差异表达miRNAs的靶基因共有549个；鉴定出8916个差异表达的基因（包括7910个保守基因和1006个新基因），在KEGG通路分析中发现一条植物-病原体相互作用的通路，共包含102个基因（1包括100个已知基因和2个新基因）。利用miRNA-seq与RNA-seq联合分析法筛选出青稞抗病相关的候选靶基因有10个（包括7个功能基因和3个转录因子）；利用功能基因家族分析法筛选出青稞条纹病相关的候选靶基因有7个（包括4个功能基因和3个转录因子）。利用降解组测序的方法对前文筛选的10个miRNAs和14个候选靶基因进行了靶向验证。最终筛选出4个miRNAs（hvu-miR168-5p、hvu-novel-11、hvu-novel-46和hvu-miR159b）和4个靶基因（AGO1、At5g45490、WRKY1和WRKY30）存在靶向关系。通过克隆获得了青稞条纹病相关的4个靶基因的CDS序列，发现在两个品种中他们的CDS区域内蛋白序列无差异。进行了转HvtAGO1基因拟南芥中条纹病的相关miRNAs和靶基因的定量表达研究，发现HvtAGO1在调控下游miRNAs和靶基因起到关键性的作用。本研究结果为今后更深入的开展青稞的抗条纹病调控机制研究奠定基础。