宝岛蕉防卫相关miRNAs介导的抗枯机制研究及香蕉抗枯种质创新

Fusarium wilt of banana caused by F. oxysporum f. sp. cubense (Foc) is one of the most severe diseases in banana and is also one of the major limiting factors for banana production in the world. We have cultured a banana mutation with wilt defence and called "baodao" banana, which have been widely planted in the South China. The transcriptome sequencing and miRNA Illumine sequencing showed that some miRNAs are differentially expressed between the "baxi" and "baodao" banana under Foc infection, and thus we supposed that a miRNA-dependent anti-fungi defense mechanism might be existed in banana plants. We plan to further elucidate the function and regulatory mechanism of miRNAs response in banana under Foc infection, and reveal whether the defence-relative miRNAs could enhance the transgenic banana plants resistance to the disease using established embryogenic cell suspensions tranformation. Recent studies have shown that the expression of silencing constructs in plants designed on fungal genes can specifically silence their targets in invading pathogenic fungi. We address important aspects for the development of fungal-derived resistance through the expression of silencing constructs in host plants as a powerful strategy to control fungal disease, and the silencing of fungicide target has provided convincing practical solutions in terms of efficiency and reliability under agronomical practice. Thus, an effective in vitro synthetic siRNA fungal screening assay was developed for screening and identifying the function of candidate genes and targets of broad-spectrum Fusarium fungicide. The lyophilized siRNAs were resuspended in the siMax Universal buffer used for in vitro Fusarium growth inhibition assay. Results of the study would provide the target gene for the HIGS that has potential for culture the resistance cultivars.

香蕉枯萎病是毁灭性的真菌土传病害，培育抗性品系是防治的关键措施，利用寄主诱导的基因沉默（Host-induced gene silencing, HIGS）在转基因植物中表达靶向病原菌的 RNAi沉默载体是培育抗病新品系的有效途径。项目组选育获得了具有一定香蕉枯萎病抗病性的"宝岛蕉"，通过转录组和差异miRNA测序后推测"宝岛蕉"存在一种依赖香蕉内源miRNA的防卫应答机制。本项目研究宝岛蕉miRNA在调控根部应答香蕉枯萎病菌侵染中的作用，揭示内源miRNA介导的抗性机制，为后续开发抗枯香蕉新种质提供理论基础。其次，以农用杀菌剂在病原真菌的作用位点作为HIGS靶标，通过转基因培育抗病品系已经有成功的报道，本项目拟通过人工合成siRNA体外筛选杀菌剂作用靶标，鉴定其在香蕉枯萎病菌中的功能并评估作为HIGS转基因抗枯的可行性，为培育香蕉抗枯新品系提供新的靶标选择和实施途径。

国家自然科学基金面上项目“宝岛蕉防卫相关 miRNAs 介导的抗枯机制研究及香蕉抗枯种质创新（31471738）”工作进展顺利，从香蕉内源免疫小RNA介导的寄主抗性机制入手，探索开发小RNA抗性资源培育抗性香蕉种质的可行性，此外，探索杀菌剂的作用靶标，评估其作为 HIGS 靶标培育抗病品系的潜在可能。.第一部分香蕉免疫小RNA相关研究。前期研究主要是对病原菌侵染后植物内源miRNA的应激表达，通过调控NBS-LRR等抗性基因的识别病原菌的效应蛋白从而诱发ETI免疫反应。我们通过Foc4人工接种水培香蕉后小RNA测序分析后证实香蕉免疫小RNA可能不参与NBS-LRR的抗性途径。因此我们马上调整实验方向，通过感染Foc4的香蕉发病组织重新分离Foc4后试图分析侵染状态下病原菌的真实小RNA表达水平，我们发现Fusarium-香蕉的分子互作中受到枯萎病侵染上调表达內源miRNA，并将miRNA导入病原菌体内特异性沉默病原菌致病基因阻止病原菌的入侵。从感病后显示症状的巴西蕉、宝岛蕉和粉蕉的蕉头重新分离纯化出病原菌Foc4进行小RNA检测，证实寄主的免疫小RNA（miR166，miR159，miR319等）可以进入到病原菌体内。这些结果证实双向跨界传导机制是一种自然存在的保守的寄主防御机制，说明小RNA在互作双方是双向传导的。我们进一步对miRNA的靶标及其功能正反分析，通过基因敲除的方法敲除miRNA的靶标基因，以及人工milRNAs技术体系人工模拟自然机制导入香蕉免疫miRNA，正反结合分析miRNA在分子互作中的机制。.第二部分是Foc4中杀菌剂靶标的鉴定及其功能初步分析。我们分别对氟吡菌酰胺，多菌灵，氰烯菌酯等杀菌剂进行平板测试，初步筛选对Fusarium有效果的杀菌剂。已经有很多研究显示β-tubulin是多菌灵的作用靶标，β1-tubulin基因敲除后生长明显受到抑制，这个结果说明β1-tubulin可以影响Foc4的生长；随着多菌灵浓度的上升，生长抑制情况更加明显，这个结果说明多菌灵可能抑制Δβ1-tubulin以外的其他基因，具有多个可能的作用位点或途径。此外，我们通过转录组测序发现了氰烯菌酯的靶标myosin-1，敲除后表型非常明显，不产孢，基本丧失了致病力，是HIGS的良好靶标选择。