玉米miR169i-p5及其靶基因ZmNuC调控粗缩病抗性分子机制解析

Maize rough dwarf disease (MRDD) is a viral maize disease that occurs worldwide, which is primary caused by the Rice black-streaked dwarf virus (RBSDV). Mapping of resistance gene and physiological and biochemical mechanism of MRDD have been extensively reported. The function of miRNA in regulating disease resistance has been recently becoming a hot area in plant. In our previous study, we have found that miR169i-p5 and its target gene ZmNuC are involved in the resistance to RBSDV infection in maize. However, its associate dregulatory mechanism has not been reported. In this project, the accurate cleavage site of target gene ZmNuC of miR169i-p5 will be identified. The overexpression vectors of miR169i-p5 and ZmNuC with mutation of target site, and CRISPR vectors of miR169i-p5 and ZmNuC will be constructed and transformed into the maize inbred line B73. Temporal and spatial expression of miR169i-p5 and target gene ZmNuC, protein expression and modification of ZmNuC will be analyzed in the functional transgenic lines. The regulatory network and resistance mechanism of miR169i-p5 mediated target gene ZmNuC of MRDD will be investigated by transcriptome, histo-cytologic approaches and their interacting genes in maize. The results obtained will not only help us to understand the theoretical basis underlying the molecular mechanism of maize resistance to MRDD, but also provide the genetic evidence and resources for breeding resistant maize germplasm.

玉米粗缩病是由水稻黑条矮缩病毒（RBSDV）引起的世界性病毒病害，虽然国内外对其抗性机制进行了广泛研究，但主要集中于抗性基因的挖掘和生理生化机制等方面。目前，miRNA调控植物抗病性功能研究已成为一个新的热点。申请者前期研究发现玉米miR169i-p5及其靶基因ZmNuC是响应RBSDV的重要调控因子，但该miRNA介导靶基因调控玉米抗粗缩病的分子机制尚不清楚。本项目拟以玉米miR169i-p5及靶基因ZmNuC为研究对象，明确其准确切割位点；分别构建miR169i-p5及ZmNuC的过表达和敲除载体，转化玉米自交系B73；选取功能明确的转基因株系分析miR169i-p5和ZmNuC在转录和翻译水平的表达及蛋白修饰；结合转录组测序、ZmNuC与玉米中基因的互作关系和组织细胞形态变化，阐明miR169i-p5介导靶基因ZmNuC调控玉米抗粗缩病的分子机制，为改良玉米粗缩病抗性提供理论基础。

玉米粗缩病（Maize rough dwarf disease，MRDD）是一种世界性病毒病害，在我国主产区由水稻黑条矮缩病毒（Rice black-striked dwarf virus，RBSDV）引起。前人在玉米bin8.03区域定位并克隆了一个隐性抗性基因ZmGDIα，其与RBSDV编码蛋白P7-1互作调控抗病性。本项目组前期结合miRNA、降解组和转录组联合分析发现玉米miR169i-p5及其靶基因ZmNuC是参与玉米粗缩病调控的重要因子，但分子机制尚未解析。本研究以玉米miR169i-p5及其靶基因ZmNuC为研究对象，利用5’RACE法证实miR169i-p5对靶基因ZmNuC的切割位点为基因3182-3200bp区域的AGGAGGAAGAAGAGGAAGA，明确了ZmNuC基因亚细胞定位于玉米细胞核，miR169i-p5及靶基因ZmNuC启动子驱动其在叶片表达，构建了miR169i-p5过表达及抑制表达载体，ZmNuC靶位点突变的过表达和CRISPR编辑载体，转化玉米自交系B104并筛选到遗传稳定的T2代阳性株系，通过玉米粗缩病人工接种鉴定和表达模式分析等揭示了miR169i-p5介导靶基因ZmNuC可提高玉米粗缩病抗性，结合ChIP-seq和LUC的方法筛选并验证了ZmNuC与编码捕光叶绿素a/b结合蛋白Zm00001d005814相互作用，ZmNuC与下游调控基因的启动子结合并抑制其表达，结合玉米与RBSDV转录水平互作关系、蛋白水平修饰、激素水平调控和细胞学水平联合分析，阐明了在玉米受RBSDV侵染后，miR169i-p5介导靶基因ZmNuC启动相关抗性机制，首先激活了茉莉酸信号转导通路，显著提高了赤霉素含量，同时ZmNuC调控下游基因Zm00001d005814影响玉米叶绿体膜和形态，参与相关光合作用进而提高其抗病性。本研究结果可为玉米粗缩病抗性机理解析和调控网络构建提供理论基础。