OsmiR1861在水稻白叶枯病抗性中的功能及分子机制

Rice bacterial blight, caused by Xanthomonas oryzae pv. Oryzae (Xoo) is one of the most serious rice diseases worldwide. Genome-wide gene expression profiles were analyzed using cDNA microarray, and three potential miRNAs were predicted using bioinformatic methods. The results of real-time quantative PCR showed that OsmiR1861k were induced in many rice varieties by different Xoo strains, and the expression of its two target genes was anti-correlated. In this study, the dynamic profiles of pre-OsmiR1861k and its target genes will be analyzed in response to Xoo. OsmiR1861k cleavage site within the target transcript will be precisely determined. Several vectors including overexpression of pre-OsmiR1861k, short tandem target mimic of OsmiR1861k and overexpression of target genes with splice-site mutation, will be constructed. Transgenic rice pure lines can be used to evaluate Xoo resistance, to analyze expression patterns of OsmiR1861k and its target genes. Therefore, we are to explain the molecular mechanism of OsmiR1861k-mediated resistance to Xoo in rice. There are no any reports about biological function of miR1861 up to now. The results will not only be useful to understand the mechanism of miR1861k on Xoo-resistance, but also provide an important opportunity for improving disease resistance of crop by miRNA genetic transformation.

革兰氏阴性菌黄单胞杆菌水稻变种(Xoo)引起的白叶枯病是水稻三大病害之一。用生物信息学方法分析已有全基因表达谱芯片结果,预测出3个miRNAs受Xoo诱导，从中选择OsmiR1861为研究对象，实验证实多个水稻品种的OsmiR1861k受多个Xoo小种诱导，并且2个靶基因的表达呈负相关。本项目将分析OsmiR1861k前体及靶基因的时序组织表达，确定对靶基因的精确切割位点，同时构建pre-OsmiR1861k 超表达、OsmiR1861k的STTM干扰及剪切位点突变的靶基因超表达载体，获得纯合转基因株系，通过测定抗病性指标、分析OsmiR1861k及其靶基因表达，探讨OsmiR1861k 调控白叶枯病抗性的功能及分子机制。目前尚无miR1861家族生物学功能的任何报道，本研究不但在水稻抗Xoo分子机理、miR1861功能方面具有重要理论意义，还可为遗传操作改良水稻抗病性提供材料。

在接种白叶枯病菌Zhe173后，OsmiR1861k成熟体和前体在抗病品种SH5中表达极显著上调，而且明显高于感病品种8411，而pre-OsmiR1861e和pre-OsmiR1861m在2个品种中均无明显上调，说明只有OsmiR1861k参与到水稻对白叶枯病的抗性。8个不同水稻品种接种Zhe173后，OsmiR1861k在敏感的3个水稻中的表达无明显变化或者显著下调，在中抗及高抗的5个基因型中显著上调。多个白叶枯病菌菌株侵染后，OsmiR1861k在抗病品种SH5中表达极显著上调，且显著高于敏感品种8411。说明OsmiR1861k在抗病品种中受白叶枯病菌诱导上调不具有品种和小种特异性。分析预测的OsmiR1861k靶基因的表达，筛选得到2个候选靶基因（Os03g18710和OsPIN2），利用RLM-5’RACE方法确定了靶基因的剪切位点。通过农杆菌介导法成功获得了3个OsmiR1861k启动子-GUS超表达、8个pre-OsmiR1861k超表达、5个STTM沉默、Os03g18710超表达和OsPIN2超表达各5个阳性株系。OsmiR1861k超表达株系中2个靶基因的表达量与OsmiR1861k表达量呈负相关，进一步验证了Os03g18710和OsPIN2是OsmiR1861k的靶基因。对各转基因3个株系与野生型SH5的叶片分别接种白叶枯病菌P3、P6小种，病斑长度及细菌生长结果显示，OsmiR1861k超表达株系的抗病性与其表达量呈正相关性；OsmiR1861k STTM及2个靶基因超表达株系的抗病性比野生型显著下降，pin2突变体的抗病性则大于野生型。3个不同抗性水稻接种P6，检测OsPIN2和6个生长素合成相关基因的表达，中抗SH5和高抗IRBB21水稻中基因AAO1、AAO2、AAO3、NIT1的相对表达量下降或者变化不大，但感病IR24水稻中这4个基因的表达量显著上升，且4个基因在抗性不同3个水稻中的表达变化与其中的OsPIN2基因表达变化趋势大致相同。推测叶片中激素IAA的合成加速协同OsPIN2活性上升导致水稻对白叶枯病表现出感病病。本项目首次确定了OsmiR1861k的生物学功能，也是少数涉及调控水稻白叶枯病抗性的OsmiRNA功能及机制的研究。