甘蔗黑穗病菌有性配合与致病相关基因SsKpFt的克隆及功能分析

Sugarcane (Saccharum spp. hybrid) is an important economic crop for crystal sugar production. Sugarcane smut disease, caused by Sporisorium scitamineum, is a major disease of sugarcane worldwide. It is also considered to be a kind of sugarcane disease with the most serious economic losses in China. S. scitamineum with different mating type spores, only two complementary mating type spores by sexual mating to form dikaryotic mycelium can develop for sugarcane smut. Therfore, it is necessary to study sexual mating mechanisms of S. scitamineum for developing new disease control strategies based on blocking or jamming sexual mating of S. scitamineum. In this study, we will clone SsKpFt gene, a mutation gene from mutant SsM23717 with defect in sexual mating and pathogenicity obtained by Agrobacterium tumefaciens-mediated transformation(ATMT) for S. Scitamineum in our laboratory, analyze the function of the gene using molecular biology methods, such as gene knockout, gene complementation, transcriptome analysis, to reveal the effects and regulation mechanism of this gene on sexual mating and pathogenicity in S. Scitamineum. The research results provide a theoretical basis for developing new disease control strategies and resistance breeding.

甘蔗是重要的糖料作物，由甘蔗鞭黑粉菌（Sporisorium scitamineum）引起的甘蔗黑穗病是世界性重要甘蔗病害，也是我国经济危害性最严重的甘蔗病害。甘蔗黑穗病菌具有不同配合型的担孢子，两个亲合的担孢子萌发后经过有性配合形成双核菌丝，才能在甘蔗体内生长蔓延及致病。因此，研究担孢子有性配合机制将有助于发展基于阻断或干扰甘蔗黑穗病菌有性配合防治甘蔗黑穗病的新型防治策略。本申请拟对项目前期利用根癌农杆菌介导甘蔗黑穗病菌遗传转化获得的一个甘蔗黑穗病菌有性配合与致病缺陷T-DNA插入突变体SsM23717的突变基因SsKpFt进行克隆，应用基因敲除、基因互补、转录组分析等分子生物学手段分析该基因的功能，揭示该基因对甘蔗黑穗病菌有性配合与致病的影响及调控机理。研究结果为发展甘蔗黑穗病新的防治策略和抗病育种提供理论依据。

甘蔗是重要的糖料作物。由甘蔗鞭黑粉菌（Sporisorium scitamineum）引起的甘蔗黑穗病是一种世界性甘蔗病害，经济损失严重，缺少有效防控措施。从分子水平揭示甘蔗鞭黑粉菌致病机理有利于发展新的防控靶标。通过对甘蔗鞭黑粉菌T-DNA插入突变体有性配合缺陷表型筛选、侧翼序列扩增分析筛选候选基因或致病力差异菌株转录组测序数据分析，筛选差异表达基因。本研究筛选出一个编码OTU1-去泛素化酶的差异表达基因。采用PEG介导的原生质体转化技术对该基因进行敲除转化及回补转化，经鉴定成功获得其阳性敲除转化子及回补转化子，并对敲除转化子、回补转化子及其野生型进行生长、形态、非生物胁迫、有性配合、致病力等表型比较及基因表达水平分析，结果表明该基因对甘蔗鞭黑粉菌非生物胁迫、细胞壁完整性、菌株生长、形态等几乎无影响，而与甘蔗鞭黑粉菌的有性配合及致病力有关。突变体间或突变体与野生型间较野生型间或回补体间或野生型与回补体间有性配合能力及致病力明显减弱，外源添加5mM cAMP或0.02mM 色醇等甘蔗鞭黑粉菌有性配合所需小分子信号物质可使突变体间或突变体与野生型间有性配合能力得到恢复。另，有性配合过程中，突变体间的色醇、cAMP合成相关基因的表达水平显著低于野生型间，而回补体间恢复至野生型水平。推测该基因可能通过调控甘蔗鞭黑粉菌有性配合小分子信号物质（cAMP或色醇）的合成参与甘蔗鞭黑粉菌的有性配合及致病力发展。本研究为甘蔗鞭黑粉菌致病机理研究提供了分子基础，为甘蔗黑穗病防控提供了新的靶标基因。