香蕉枯萎菌侵染过程中编码microRNA-likes介导的基因调控机制研究及抗枯种质创新

Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense (Foc) is one of the most severe diseases in banana and host-induced gene silencing (HIGS) is a new strategy for development the new anti-wilt germplasm. We have attempted to control the Fusarium wilt of banana by rotating and intercropping with Chinese chive (Allium tuberosum Rottler) and Chinese leek, supplying silicon (Si) to banana plants and biological controlling, but failed to effective control the disease. . MicroRNAs (miRNAs) are small RNAs that play important roles in the regulation of gene expression. It has recently been reported that many virus, most with DNA genomes, but at least some with RNA genomes, encode miRNAs or microRNA-likes (milRNAs). These regulatory sRNAs facilitate infection by suppressing host target genes involved in a wide range of key cellular pathways. Accordingly, bacteria could utilize endogenous noncoding RNAs to affect host physiology by targeting host gene. Furthermore, the discovery of fungal sRNAs that are delivered into host cells to suppress plant immunity added sRNAs to the list of pathogen effectors. Therefore, the sRNA can act as mobile sRNA signaling molecules signaling that trans-kingdom transfer and trigger cross-kingdom gene silencing.. We have differential analysis the small RNA database between media cultured Foc and artificial inoculum water-cultured “baxi” banana, the result showed that Foc could encode microRNA-likes (milRNAs) and microRNA-like siRNAs during infection process, supposing the Foc could produce small RNA facilitates replicate. Furthermore, the most of identified microRNA-likes could predict targets in banana transcription. However, the precise contribution that Foc-encoded microRNA-likes make to phytopathogenic fungi during infection process and pathogenesis in vivo is unknown. . To elucidate the regulatory mechanism of Foc-encoded microRNA-likes facilitate infection and target host gene in a trans-kingdom manner, we use previously established GFP-tagged artificial microRNA-like technique and water-cultured banana platform to explore the candidate microRNA-likes that involved in pathogenicity. Then, the expression profile of candidate milRNA and the relationship between the predicted target gene in Banana host. Furthermore, we also evaluated the feasibility that targeting pathogenicity-related microRNA-like could confer the transgenic banana plants resistance to the disease using established embryogenic cell suspensions transformation, using previously established Fusarium oxysporum artificial milRNA technique combination with water-culture banana platform. We address important aspects for the development of Foc encoded milRNA-derived resistance through the expression of silencing constructs by targeting Foc-encoded pathogenesis-related microRNA-likes in host plants as a powerful strategy to control fungal disease.

香蕉枯萎病（Foc）是毁灭性的土传病害，培育抗枯品系是防治的根本途径。小RNA测序已经证实Foc在侵染过程中产生自身编码microRNA-likes（milRNAs），并且可以在香蕉的转录本中预测到大多数milRNAs的靶标基因，推测Foc在侵染寄主过程中存在一种依赖自身编码milRNAs介导的基因调控机制促进侵染。本项目拟在青年基金研究的基础上，利用开发的人工milRNA基因沉默技术体系，构建候选milRNA过量表达、抑制表达的载体遗传转化Foc后筛选稳定表达的转化子，人工接种水培香蕉，分析候选milRNA及其靶标的相关性，从正、反两方面研究milRNAs在Foc侵染过程中的作用，揭示Foc依赖自身编码milRNA介导基因调控从而促进侵染的分子机制。通过评估milRNAs作为RNAi基因沉默靶标HIGS转基因抗枯的可行性，为培育香蕉抗枯品系提供新的靶标选择和实施途径。

香蕉枯萎病（Foc）是毁灭性的土传病害，培育抗枯品系是防治的根本途径。小RNA测序已经证实Foc在侵染过程中产生自身编码microRNA-likes（milRNAs），并且可以在香蕉的转录本中预测到大多数milRNAs的靶标基因，推测Foc在侵染寄主过程中存在一种依赖自身编码milRNAs介导的基因调控机制促进侵染。本项目拟构建候选milRNA过量表达、抑制表达的载体遗传转化Foc后筛选稳定表达的转化子，人工接种水培香蕉，分析候选milRNA及其靶标的相关性，从正、反两方面研究milRNAs在Foc侵染过程中的作用，揭示Foc依赖自身编码milRNA介导基因调控从而促进侵染的分子机制，为培育香蕉抗枯品系提供新的靶标选择和实施途径.本项目实际研究结果均低于项目预期目标。目前发表7篇论文，申请并授权专利5项，编写 “香蕉枯萎病综合防控理论与实践”专著1本，获得香蕉新品种权“热科2号”1项，获得海南省人才项目“南海名家”1项目，整个团队获得海南省高层次人才称号5项，项目执行期间培养了3名硕士研究生，其中2名已经毕业。但是，目前核心的实验没有完成，小RNA的功能鉴定工作并未完成。.此外，项目还取得一些与当初设想存在差异的研究结果：1）通过基因敲除的方法或的枯萎菌AGOs和DCLs缺失突变体，通过小RNA测序确定了他们在小RNA生物合成途径的作用，以及对致病力的影响；明确了枯萎菌中AGO和DCL基因并不是小RNA产生的核心基因，它们对致病力的影响也不明显。2）枯萎菌中富含RNaseIII功能域的基因可能是小RNA产生的核心基因，也是枯萎菌必需的基因，决定了枯萎菌的致病力。3）枯萎菌致病相关小RNA的鉴定及其敲除实验，证明敲除单一的Foc4小RNA后致病力仍然显著下调，说明小RNA在枯萎菌的侵染中发挥重要作用。