利用CRISPR/Cas9技术筛选苜蓿镰刀菌根腐病抗病与感病基因

Alfalfa Fusarium root rot is a potential and chronic devastating disease in alfalfa-growing areas worldwide. Alfalfa Fusarium root rot is soil-borne in nature, as long as pathogens of Fusarium species present in the soil and the condition suitable, the disease can infect alfalfa in all development periods. Root rot injured as characterized by blackish-brown lesions appearing on root surfaces, then taproot and lateral root gradually decay; The number of new shoot was decreased in severely affected crowns; Nitrogen fixation declined. As there is no chemical and management can cure this disease permanently, the majority of the fields show a progressive deterioration after the second year, yield and quality also effected greatly. At present, the root rot is one of limiting factors to alfalfa production throughout the world and is difficult to control. Therefore, the breeding of resistant cultivar is the most economical and environmentally friendly method for controlling root rot disease, while understand and screening of alfalfa resistance and susceptibility genes is the fundamental for rapid breeding disease-resistant cultivar. As of today, which and how many gens were involves in resistance and susceptibility have not been discovered, the molecular mechanisms that contribute to pathogenesis and defense responses in alfalfa against Fusarium spp. still unclear. Here, we try to analyze gene regulatory network and then screen resistance/susceptible genes. Therefore, based on single-molecule long-read sequencing and the second-generation transcriptome sequencing, obtain alfalfa full-length transcriptome sequence and resistance/susceptible gene pool. Then, identify and screen resistance/ susceptible genes from up/down regulate significantly genes by CRISPR/Cas9 genome editing system. Our project will lead to important strategies for research on disease resistance of alfalfa Fusarium root rot and will provide gene candidates for breeding alfalfa cultivar which can resistant to Fusarium root rot.

苜蓿镰刀菌根腐病是世界上发生较为广泛的病害之一，普遍发生在各个苜蓿栽培地区。因其土传性，只要条件适合，植株发育的各个时期均可感病。感病后，根颈和主根木质部逐渐腐烂中空，侧根大量死亡，分枝减少，固氮能力下降，无法通过药物和田间管理措施根治，导致利用年限缩短，草产量和品质降低。目前，该病害已经成为苜蓿大面积推广的一个主要限制因素。因此，抗病品种的培育显得尤为重要，而筛选和研究苜蓿的抗病和感病基因是高效快速培育抗病品种的基础。但目前为止，苜蓿镰刀菌根腐病的调控网络还未进行全面地研究，抗病和感病基因的鉴定筛选工作更是一片空白。因此，本项目拟通过第三代单分子全长转录组测序与二代转录组测序相结合，获得苜蓿全长转录组序列，并通过qRT-PCR分析建立抗病/感病基因池，在此基础上采用CRISPR/Cas9技术对上调/下调显著的基因进行鉴定，筛选出抗病和感病基因，为苜蓿镰刀菌根腐病分子抗病育种提供候选基因。

紫花苜蓿（Medicago sativa L.）是全世界最重要的豆科牧草之一，随着种植模式的改变和种植面积的增加，由镰刀菌引起的根腐病已成为影响其产量和品质的一个重要因素。因此，抗病品种的培育显得尤为重要，而筛选和研究苜蓿的抗病和感病基因是高效快速培育抗病品种的基础。但目前为止，苜蓿镰刀菌根腐病的调控网络还未进行全面地研究，抗病和感病基因的鉴定筛选工作更是一片空白。因此，本项目中将筛选的抗病单株和感病单株接种层出镰刀菌（Fusarium proliferatum）后27h、48h、72h和7d取样进行转录组测序，在生新分析和qRT-PCR结构的基础上，建立抗病/感病基因池，并通过过表达和CRISPR/Cas9基因编辑手段对候选基因进行功能鉴定。根据试验结果，病程相关基因非表达子（Nonexpresser of pathogenesis-related genes, NPR）在植物系统获得性抗性中有着不容忽视的重要性及研究价值。另外，在抗性株系中发现了一个有趣的现象: 神秘果素（miraculin）基因和多聚半乳糖醛酸酶抑制剂基因（polygalacturonase inhibitor gene）在接种初期表达量极显著上调，初步确定这两个基因在参与抗病过程有很重要的功能。本项目筛选的抗病和感病基因可为苜蓿镰刀菌根腐病分子抗病育种提供候选基因。