茎瘤芥根肿菌侵染分子生态机制研究

Clubroot disease is one of the major diseases in the production of cruciferous crops, which seriously restricts the sustainable development of the mustard industry. The resting spores of Plasmodiophora brassicae lived in soil for a long period of time and were the main primary source of infection. The germination and infection of resting spores were stimulated by host root exudates. This project intends to inoculate tumorous stem mustard cultured in solution with resting spores of P. brassicae, to investigate the stimulatory effects of roots on the germination and infestation of P. brassicae. Further analysis of transcriptome and proteomics based on genome data to screen of infection-related genes of P. brassicae induced by roots and to confirm their signal transduction pathways. Then, siRNA-based RNAi technology was used to analyze the function of the genes and combined the germination and infection phenotypes to screen the key infection-related genes. Finally, nanomaterials were used to transport complex siRNA to analyze the effect of the mixed siRNA on the germination, infection and virulence of P. brassicae, and the optimal compound siRNA system was screened in combination with target gene expression data. Through the research of this project, the molecular ecological mechanism of the germination and infestation of P. brassicae resting spores in response to tumorous stem mustard roots can be clarified, and the core genes that play a key role in this process can be identified, and the use of nanomaterials to transport complex siRNA can provides a theoretical basis and technical support for the development of "bio-pesticides" to control clubroot disease.

根肿病是十字花科作物生产的主要病害之一，严重制约榨菜产业的可持续发展。根肿菌休眠孢子在土壤中存活时间长，是主要的初侵染源，受寄主根系分泌物刺激后萌发侵染。本项目拟在液培茎瘤芥中接种根肿菌休眠孢子，探索茎瘤芥根系对其萌发和侵染的刺激作用，进一步进行转录组和蛋白组分析，结合基因组数据筛选茎瘤芥根系诱导的根肿菌侵染相关基因，明确基因的信号转导通路。再通过基于siRNA的RNAi技术，分析根肿菌侵染相关基因的功能，结合萌发和侵染表型，筛选侵染关键基因。最后利用纳米材料转运复配siRNA，分析复配siRNA对根肿菌萌发、侵染和致病力的影响，结合靶标基因表达数据，筛选最适的复配siRNA体系。通过本项目研究可阐明根肿菌休眠孢子响应茎瘤芥根系刺激后萌发侵染的分子生态机制，明确该过程中起关键作用的侵染核心基因，而利用纳米材料转运复配siRNA则可为开发“生物农药”防治根肿病提供理论依据和技术支撑。

根肿病是影响茎瘤芥生产的主要病害之一，目前仍以化学农药防治为主。根肿菌侵染茎瘤芥的分子机制未知，目前已明确根肿菌中部分效应子基因是其侵染的主要工具，此外海藻糖是根肿菌抵御不良环境和萌发侵染的能量来源。本项目利用茎瘤芥根系分泌物处理根肿菌后，通过转录组和蛋白组测序筛选到差异表达基因219个，其中效应子基因8个，预测的真菌致病基因13个。此外，对根肿菌侵染茎瘤芥不同时期的效应子基因和海藻糖合成和代谢基因进行表达模式分析。由此，从中选择20个在根肿菌侵染茎瘤芥不同时期起作用的效应子基因和5个海藻糖合成和代谢基因作为siRNA处理的靶标基因。在这25个基因中，目前筛选到靶向6-磷酸海藻糖合成酶基因（PbTPS1）的1对siRNA能显著降低茎瘤芥根肿病发病程度，靶标基因表达水平下降约57%，处理后肿根中海藻糖酶活和海藻糖含量显著降低，显著抑制肿根形成，但该siRNA对根肿菌休眠孢子萌发影响不显著；靶向根肿菌效应子基因PBRA7977、PBRA7592和PBRA6928的混合siRNA能显著促进根肿菌的侵染，这3个基因在休眠孢子中表达水平较高，但在侵染过程中表达水平较低，推测这3个基因的表达可能激活植物的防御反应，因此利用siRNA处理后能促进根肿菌的侵染；靶向根肿菌效应子基因PBRA3510的siRNA在根毛侵染中能显著下调靶标基因表达，对茎瘤芥肿根形成的影响需进一步探索。最后，我们从8种纳米材料中筛选到3-Mg-200和1-MgO对根肿病具有较好的防效，作为后期搭载siRNA的候选纳米材料。综上所述，根肿菌PbTPS1对根肿菌侵染茎瘤芥和合成海藻糖非常重要，根肿菌效应子基因PBRA7566、PBRA7977、PBRA7592、PBRA6928可能在根肿菌侵染茎瘤芥过程中激活寄主的防御反应，不利于根肿菌的侵染。筛选到的2种纳米材料则具有较好的田间应用价值。这些结果为深入探索根肿菌侵染茎瘤芥的分子机制和利用纳米材料防治根肿病奠定了基础。