西瓜枯萎菌人工miRNAs基因沉默技术构建及致病相关基因鉴定分析

西瓜枯萎病是由尖孢镰刀菌引起的一种世界范围内的真菌性土传病害。有文献表明，通过基因诱导的基因沉默（Host-induced gene silencing，HIGS）在寄主中表达RNAi沉默载体是培育抗病原真菌的新品系的有效措施。 microRNAs是一类真核生物广泛存在的重要基因表达调控因子。目前，依据microRNAs骨架序列构建的人工miRNAs基技术广泛应用在植物功能基因分析中。真菌中虽然存在与植物microRNA类似的小RNA，但目前还未建立人工miRNAs技术。课题组前期测定了西瓜枯萎菌小RNA表达谱，初步试验表明，PEG介导的原生质体转化小RNA骨架序列可以体内加工成为成熟的小RNA。 本项目模仿植物人工miRNAs的原理和方法，构建尖镰孢西瓜转化型的人工miRNAs基因沉默技术，并验证西瓜枯萎菌突变体库中的候选致病相关基因功能，为后续采用HIGS培育抗性品系提供理论依据。

Fusarium wilt of water-melon caused by F. oxysporum f. sp. niveum(E.F. Smith) Snyder &Hansen (Fon) is one of the most severe diseases in watermelon and is also one of the major limiting factors for watermelon production in the world. 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, such as Fusarium verticillioides, Blumeria graminisand Puccinia striiformis f.sp. tritici.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. The artificial microRNA (amiRNA) technology exploits endogenous miRNA precursors to generate sRNAs that direct gene silencing in either plants or animals. Although the plant microRNAs-like small RNAs existed in fungi, the artificial microRNAs technique is not established till now. Consequently, we have study the small RNA expression profile of Fon with illumina deep sequencing plantform, and have proved that the small RNA precursor of Fon could generate the mature miRNA by PEG-mediated transformation. This study was aim to construct the artificial miRNAs platform according to the principle and method of plant miRNAs, and verify the pathogenicity-relative candidate gene in the T-DNA insertional library of Fon using the gene silencing technique. Results of the study would provide the target gene for for the HIGS that has potential for culture the resistance cultivars.