gma-miRNA1510调控大豆疫霉根腐病抗性的作用机制研究

microRNAs (miRNAs) have been shown to play a role in plant resistance by targeting mRNAs that code for the auxin receptors in Arabidopsis and other plants. However, whether the miRNAs function in the interaction between soybean and Phytophthora sojae (P. sojae) is unknown. Expressional patterns of miRNAs upon infection by P. sojae by microarray analysis were obtained in three soybean cultivars (Williams 82, Conrad and Williams), and we found that the targets of gma-miR1510 are involved in plant defence. We inferred that gma-miR1510 influence soybean resistance to P. sojae. In this research, we adopt RLM-RACE and GFP-dependent method to validate the interaction between gma-miR1510 and its predicted targets in vivo. Putative targets are fused to the 5' end of GFP and introduced into Nicotiana Benthamiana (N. benthamiana). Leaves from transgenic N. benthamiana are infiltrated with A. tumefaciens harboring the 35S promoter driven gma-miR1510 precursor. Then, we will generate gma-miR1510 overexpressing and targets silencing transgenic soybean plants by Agrobacterium-mediated transform system and analyze miRNA functions through resistance and cellular oberversation on transgenic plants. This will help to understand the mechanism of gma-miR1510 regulation of soybean resistance to P. sojae, and provide new materials for resistance breeding of soybean.

最近的研究表明microRNA(miRNA)参与调控植物的抗病性，但miRNA在大豆抗疫霉根腐病过程中的作用和机制尚未见报道。本实验室利用生物芯片技术筛选到一批受大豆疫霉菌(Phytophthora sojae, P. sojae)侵染调控的miRNAs，发现其中的gma-miR1510在不同抗性水平的大豆品种中具有不同的表达水平，生物信息学预测其多个靶标基因与植物的防卫反应相关。本研究拟通过RLM-RACE技术和基因瞬时表达技术确定gma-miR1510的有效靶标基因；然后利用大豆的遗传转化技术获得gma-miR1510的过量表达植株和对应靶标基因沉默的大豆转基因植株，并对转基因后代进行抗性鉴定和细胞学分析，研究gma-miR1510及其靶标基因在大豆抗疫霉根腐病中的功能，探索gma-miR510在调控大豆抗疫霉根腐病抗性中的作用机制，为大豆的抗性分子育种奠定理论基础并提供新的育种材料。

由大豆疫霉菌(Phytophthora sojae Kaufmann & Gerdemann，P. sojae)引起的大豆疫霉根腐病是大豆生产上的毁灭性土传病害之一。microRNA(miRNA)是真核生物中发现的一类内源性的具有调控功能的非编码小分子RNA，通过碱基互补配对的方式识别靶标mRNA，并根据互补程度的不同指导沉默复合体降解靶标mRNA或者抑制靶标mRNA的翻译。已有研究表明，miRNA参与调控植物的抗病性。利用基因芯片筛选受大豆疫霉菌调控的miRNA，发现gma-miR1510受大豆疫霉菌P6497侵染后，其表达量与对照相比显著下调。本项目主要研究内容包括gma-miR1510生物信息学分析、靶基因预测及验证、前体和有效靶标基因的启动子顺式作用元件分析、gma-miR1510过表达大豆根毛的获得及分析gma-miR1510调控大豆疫霉根腐病抗性的作用机制。本研究发现大豆受到疫霉菌侵染后gma-miR1510的表达被抑制，说明gma-miR1510可能参与大豆对病原菌的响应。通过构建人工miRNA前体的方法将gma-miR1510a/b分别在大豆发状根中过表达，发现gma-miR1510a/b过表达的发状根与空载体对照相比表现为对大豆疫霉更加感病。5’RACE实验证明gma-miR1510以直接裂解mRNA的方式调控靶基因Glyma.16G135500，该基因编码TIR和NB-LRR类型的抗性蛋白。gma-miR1510和Glyma.16G135500在疫霉菌胁迫下的表达模式呈负相关。对两者启动子的顺式作用元件进一步分析发现，存在响应生物胁迫和激素的顺式作用调控元件。这些结果表明，gma-miR1510通过调控其靶标基因Glyma.16G135500从而参与大豆对大豆疫霉菌的抗性。