MicroRNA与NBS-LRR类受体蛋白在SMV-大豆互作过程中的作用研究

Soybean Mosaic Virus (SMV) is a plant virus that infects soybean worldwide wherever soybean is grown, causing severe yield reductions. SMV can also contribute to crop loss by causing seed coat mottling thus decreasing seed quality. MicroRNAs are endogenous、 non-protein-coding small RNAs that play important regulatory roles in animals and plants by targeting mRNA for cleavage or translational repression. Plant virus infections can result in disease symptoms that may include chlorosis and/or necrosis, curling, stunting and altered plant stature and morphology, presumably caused by interference of the infection with developmental processes. In recent years, it has been proven experimentally that microRNA play important roles in plant development and are implicated in virus-induced symptom development,presumably due to the fact that the accumulation of microRNAs are influenced by the virus component proteins including suppressor protein.In soybean genome,554 mature microRNAs are reported. In the present study, the accumulation of these microRNAs would be studied as a result of the infection of different SMV species with varying virulence using high-throughput microRNA Microarray analysis. Expression of some important microRNA and their target genes,especially those involved in small RNA biogenesis and NBS-LRR disease-resistant genes would be validated by real-time qPCR, and their effector-target relationships would be confirmed. The possible role of microRNA and their target gene product in SMV pathogenicity and host-patogen interactions would be assessed in soybean.

大豆花叶病毒病（Soybean Mosaic Virus，SMV）是一种世界性病害，SMV的侵染导致大豆减产，并引起种粒斑驳等症状，对大豆的经济和商品价值有着严重的影响。MicroRNA是一类由生物体内源产生的、对其生长发育等生命过程有重要调节作用的非编码功能的小RNA分子。近年来，有一些研究表明，病毒侵染后会导致一些重要的microRNA的表达量的变化，从而引起了寄主特有的表型变化。本项研究拟利用microRNA芯片检测不同致病力SMV侵染后大豆叶片中MicroRNA表达谱的变化， 其中重点研究大豆NBS-LRR类抗病基因及 small RNA biogenesis相关基因调控有关的MicroRNA在植物对病毒侵染应答的作用。 通过上述试验我们可以初步探讨植物microRNA及其靶标mRNAs与SMV侵染的关系,为进一步揭示SMV-大豆互作的分子机制打下基础。

这是国家自然基金面上探索性一年期（2014，1-12月）项目“MicroRNA与NBS-LRR类受体蛋白在SMV-大豆互作过程中的作用研究”的结题报告。. 大豆花叶病毒病（Soybean Mosaic Virus，SMV）是一种世界性病害，SMV的侵染导致大豆减产，并引起种粒斑驳等症状，对大豆的经济和商品价值有着严重的影响。MicroRNA是一类由生物体内源产生的、对其生长发育等生命过程有重要调节作用的非编码功能的小RNA分子。近年来，有一些研究表明病毒侵染后会导致一些重要的microRNA的表达量的变化。本项研究利用microRNA芯片检测了SMV N1侵染后大豆叶片中MicroRNA表达谱的变化，共利用554个探针进行检测发现有40个 microRNA的水平发生了显著的变化，其中26个上调，14个下调。选取25个受SMV调控的microRNA进行了qRT-PCR检测，证明有24个与芯片结果一致，其中13个与NBS-LRR蛋白基因的调控有关。接着我们重点研究了5个NBS-LRR类抗病蛋白基因及4个 small RNA biogenesis相关蛋白基因调控有关的MicroRNA与其预测靶标基因在SMV侵染后的表达变化。 我们通过Northern杂交进一步证明了这9个microRNA在SMV N1 和 SMV N3侵染后的上调关系，并验证了其靶标基因的表达与其调控的microRNA具有很好的对应关系。通过上述试验我们初步探讨了大豆microRNA及其靶标mRNA与SMV侵染的关系, 完成了计划书所列研究内容，为进一步揭示SMV-大豆互作的分子机制打下了基础。另外，gma-miR-2的靶标为与烟草抗病蛋白R蛋白编码基因同源的R-homolog基因，gma-miR-2和 R-homolog基因的表达在SMV侵染后的不同时间点均表现出很强的调控关系，因此我们正在检测大豆35个R-homolog基因在SMV侵染中的作用及gma-miR-2的调控机理，这是本课题实施中发现的一个重要的现象，也是将来深入研究的一个方向。.