辣椒抗根结线虫Me3基因的克隆及其抗性分子机制研究

Root-knot nematodes (Meloidogyne spp.) are important plant-parasitic nematodes, with a worldwide distribution. They can parasitize thousands of plant species and result in devastating adverse effects on the quality and yield of crops. Host resistance is considered as the most efficient and safe strategy to control Meloidogyne spp. The new heat-stable nematode-resistance gene is an important element for durable utilization of resistant genes and crop breeding. Me3 is a single dominant root-knot nematode-resistant gene, but until now, it hasn’t been cloned. In this study, we have completed the deep sequencing of DNA pools of resistant and susceptible plants of F2 mapping population respectively, and have identified the BAC clones contigs of pepper Me3 gene. First, we plan to complete the fine mapping of Me3 and re-sequencing of Me3 gene exchange single plant and HDA149 pepper, combining the sequencing of Me3 gene BAC clone, found the candidates, then clone the Me3 gene. Second, we will analyze the Me3 gene biological functions by the CRISPR/Cas9 gene editing system and the pepper transgenic technology mediated by Agrobacterium. In addition, we will reveal that the immune response molecular mechanisms with the transcriptomes of HDA149 plant infected by nematodes, and analyze the heat-stable nematode-resistant mechanisms in thermal treatment experiments. These research results will be beneficial for vegetable heat stable nematode-resistant breeding and durable utilization of nematode-resistant genes.

根结线虫每年给全球农业生产带来了巨大的经济损失，利用抗线虫基因防治根结线虫是最经济、安全、高效的策略。新的热稳定性抗线虫基因及其抗性机理研究是抗线虫育种及抗性基因持久利用的关键。Me3基因是辣椒的单显性抗根结线虫基因，抗性广谱，且具有热稳定性,但一直未能克隆。在研究前期已完成了F2代极端抗感DNA池的构建及深度重测序，构建了BAC文库并明确了Me3基因BAC重叠群，在此基础上我们拟利用F2代作图群体对Me3基因进行精细定位，确定遗传交换区域，筛选抗性遗传交换单株并重测序，结合BAC测序确定候选基因，经鉴定及遗传验证克隆Me3基因。采用基因编辑技术及转基因技术对Me3基因进行生物功能分析。同时，通过Me3基因材料与根结线虫分子互作、转录组分析等探讨Me3基因介导的信号传导途径、免疫应答及热稳定抗性机制，这些研究对于突破抗线虫育种瓶颈及抗性基因持久利用具有重要意义。

采用抗线虫基因是防控根结线虫的安全高效策略，高效的抗线虫基因挖掘是重要研究方向。本研究通过构建F2代高密度精细图谱，定位克隆了辣椒中的热稳定性抗线虫基因Me3的候选基因Ca163，DNA全长6326bp，cDNA最大ORF为2211bp，为NBS-LRR型抗性基因结构。通过多组合F2代遗传特征分析，证实了候选基因Ca163符合单显性基因的遗传规律。通过VIGS基因敲低，番茄及拟南芥转基因超表达技术，验证了该基因具有抗根结线虫作用，进一步的说明候选基因Ca163即为辣椒中的抗线虫基因Me3。同时，基于抗感亲本中靶标区域DNA序列差异开发了SCAR分子标记SCAR537，为辅助抗线虫育种提供了依据。通过室内及温室试验证实Me3基因具有热稳定性，在35℃下仍具有稳定的抗性。通过瞬时超表达体系，证实Me3基因可以诱发过敏性坏死反应，同时根结线虫效应子Min10750与该基因互作可以抑制Me3基因诱发的HR反应，揭示了Me3基因与线虫效应子存在免疫应答反应。研究结果对于开发利用Me3基因及防控根结线虫提供了重要依据。