甘蓝与尖孢镰刀菌粘团专化型不亲和互作的机制研究

Fusarium oxysporum f. sp conglutinans (FOC), a major soil-borne fungal pathogen and one of the most destructive diseases of cabbage worldwide, causes cabbage vascular wilt disease in our country in recent years. It is significant important for the analysis on genetic effects of the cabbage resistant genes to FOC on cabbage resistant breeding. Our previous investigations have revealed the pathogenesis of FOC and the initial genetic resistance of cabbage. Moreover, we have completed the comparative genome sequencing of FOC race1 and race2. Importantly, we have finished the resequencing of pathogen collections of Chinese isolations. Based on the above results, we will plan to further conduct the following researches. Firstly, through comparing the difference transcriptomes among three genotypic cabbages (SS, RS, RR) that inoculated with race1 and race2 using RNA-Seq based on Illumina, we will screen and identify the putative important genes that might take part in the incompatible interactions between cabbage and FOC. Then we will analyze the dynamic expression of these genes during the incompatible interactions using qPCR. Next we will conduct the functional analysis by genetic transformation of these genes on susceptible cabbages. Furthermore, we will use the RNA interfering method to knockdown the expression of endogenous genes to reveal the gene functions of FOC race1 and race2. Our goal is to clarify the molecular basis of the interactions between cabbage and FOC, and the results will provide theoretical guidance for establishing innovative control methods and breeding strategy of resistant cabbages.

甘蓝枯萎病是近年在我国新发生的一种毁灭性土传病害，探索甘蓝与枯萎病菌之间的不亲和互作，对于解析甘蓝抗枯萎病基因的遗传效应，培育抗枯萎病品种具有重要的意义。本项目拟在前期初步揭示甘蓝枯萎病菌的致害机制、明确品种抗性遗传规律、完成尖孢镰刀菌甘蓝专化型1号和2号生理小种的比较基因组测序和国内分离物重测序的基础上，采用基于Illumina 的RNA-seq技术，比较三种基因型甘蓝（SS，RS，RR）在1号/2号小种侵染下的全基因组转录组差异，筛选出甘蓝与尖孢镰刀菌粘团专化型不亲和互作相关的重要功能基因，并对这些基因参与抗病的作用过程进行qPRC表达动态分析；进一步对重要基因进行感病基因型甘蓝遗传转化功能分析；同时，利用同源重组敲除方法对尖孢镰刀菌粘团专化型1号/2号小种进行基因敲除功能分析。以期阐明甘蓝枯萎病菌与甘蓝互作的分子机制，为制定具有创新性的枯萎病控制及育种策略提供理论依据。

甘蓝枯萎病（Foc）是近年在我国新发生的一种毁灭性土传病害，解析Foc侵染寄主的分子机制及在互作过程中发挥重要的基因，对于解析甘蓝抗枯萎病菌基因的遗传效应，培育抗枯萎病品种具有重要的意义。本研究首先开发了一种灵敏、高效的甘蓝枯萎病菌小种1和小种2（Foc1、Foc2）的分子鉴定方法。通过GFP标记，明确了Foc侵染甘蓝的过程，并解析了抗性品种抑制病原菌发育的方式。在此基础上，我们通过基因组，转录组和蛋白组数据来鉴定不亲和互作相关的重要功能基因。通过比较基因组，鉴定了Foc1特有4.3M片段和146个特异基因，Foc2特有3.2M片段和435个特异基因R；通过比较三种基因型甘蓝（SS，RS，RR）在1号/2号小种侵染下的全基因组转录组数据，分别鉴定了1365，876和1267个差异表达的基因，其中107个基因为SS特有，56为RR特有，98为RS特有。这些特异基因可以分类为转录因子、细胞壁修饰基因、病原物相关基因以及效应子蛋白等。差异基因的调控途径分析表明转录因子参与了这些差异基因的调控。通过Foc1和Foc2的蛋白组分析，鉴定了145个差异蛋白，进一步通过比较Foc1和Foc2菌丝和孢子的差异表达蛋白，发现参与碳转运和代谢类相关蛋白对甘蓝致病力有着显著的影响。结合上述比较基因组、转录组、蛋白组数据，我们筛选出了27个参与不亲和互作的关键效应子基因，其中一类CFEM效应子蛋白在Foc侵染寄主的过程中高度表达，敲掉CFEM后，病原菌的致病力显著下降，但生长发育不受影响，表明CFEM直接决定了Foc的致病力。进一步我们对筛选出的关键基因进行了功能研究，发现转录因子PEREGRIN直接参与了Foc1的抗氧胁迫过程；β-1, 3-葡聚糖酶对Foc侵染过程中菌丝的发育至关重要；SIX1可以直接影响Foc的致病力。对这些基因功能的研究，进一步加深了我们对病原物和寄主互作过程中的分子机制的理解。