猕猴桃果实熟腐病关键抗病相关基因筛选及功能鉴定

Fruit ripe rot of kiwifruit, caused by the fungus Botryosphaeria dothidea, is a latent infective disease mainly infected in young fruit period and shows symptoms in fruit nearly mature and postharvest period. Since the disease currently occurs widespreadly in kiwifruit growing regions of China and abroad and results in severe economic loses, it is urgent to exploit fruit ripe rot resistance genes and therefore to breed kiwifruit varieties resistant to the disease. On the basis of having screened out highly resistant, moderately resistant and highly susceptible kiwifruit varieties resistant to the disease, the three varieties will be inoculated respectively with the high pathogenicity strain BDXF-1of B. dothidea and their transcriptome sequencing will be carried out at regular time intervals using RNA-Seq technology.Function of each expressing gene will be annotated by referring to whole genomic sequence information of kiwifruit which has been reported recently. By comparison of gene expression differences among different varieties as well as different time points, the differentially expressed genes related to disease resistance will be hopefully screened out and the key disease resistance related genes will be judged. After that, the full length cDNA of key disease resistance related genes will be cloned and sequenced, and their functions will be identified. Upon completion of the project, We will not only obtain a number of resistance related genes against fruit ripe rot of kiwifruit, but also will elucidate the molecular mechanism of fruit ripe rot resistance in kiwifruit on gene transciption level.

猕猴桃果实熟腐病,由葡萄座腔菌（Botryosphaeria dothidea）引起，是一种主要在幼果期侵入而在果实近成熟期特别是在贮藏期发病的潜伏侵染性病害。鉴于该病害目前在国内外猕猴桃产区普遍发生并导致严重的经济损失，迫切需要发掘果实熟腐病抗病相关基因，培育抗熟腐病猕猴桃新品种。本项目在前期筛选到对果实熟腐病表现为高抗、中抗和高感猕猴桃品种的基础上，用葡萄座腔菌强致病力菌株BDFX-1分别对其进行挑战接种，采用RNA-Seq技术对挑战接种后不同时间点果实进行转录组测序，参考最近测序完成的猕猴桃全基因组序列信息对各表达基因进行功能注释；比较不同抗性品种及不同时间点基因表达差异，从中筛选差异表达抗病相关基因和确定关键抗病相关基因；对关键抗病相关基因进行全长克隆、序列测定和功能鉴定。本项目的完成，不仅能发掘一批猕猴桃果实熟腐病抗病相关基因，而且将在转录水平上揭示熟腐病抗性的分子机制。

由葡萄座腔菌引起的猕猴桃果实熟腐病是影响国内外猕猴桃生产的重要病害之一。本项目在前期筛选到对果实熟腐病表现为高抗和高感猕猴桃品种的基础上，用葡萄座腔菌强致病力菌株GF-27对抗病品种“金艳”和感病品种“红阳”的果实进行田间挑战接种，并设不接菌处理作对照，在接种后1d、3d、6d取样，共取样品36个；提取各样品总RNA，构建各样品的cDNA文库，使用Illumina Hiseq TM2000技术进行转录组测序，将测序结果与已发表的猕猴桃全基因组序列信息进行比对。结果从36个样品中共获得305.24Gb干净的测序数据（clean data），其中抗病品种为175.76 Gb，感病品种为12 9.48 Gb。共鉴定到44656个表达基因，其中39041个是已知基因，5615个是新基因。应对葡萄座腔菌侵染，红阳共有13910个基因发生差异表达，其中上调表达基因7300个，下调表达基因6610个；金艳共有5574个基因发生差异表达，其中上调表达基因3570个，下调表达基因2004个。采用qRT-PCR验证Illumina测序结果的准确性，结果同一个差异转录本在qRT-PCR和Illumina试验中的表达水平基本一致，表明本研究中Illumina测序结果较为可靠。参考植物病害防御相关文献，发现了2373个差异表达基因可能参与了猕猴桃对果腐病的抗病反应，根据这些基因在抗感品种中的表达差异及在抗病品种中各时间点的表达趋势，确定了30个关键抗果腐病相关基因，这些基因涉及到钙信使系统、MAPK途径、细胞壁修饰、植保素合成、转录因子、细胞表面的模式识别受体和病程相关蛋白等多方面，其中以钙信使系统、MAPK途径和细胞壁修饰基因在抗病品种中上调更加明显。选择其中的10个木葡聚糖内糖基转移/水解酶（XTH）抗病基因和1个几丁质酶基因进行克隆和生物信息学分析，获得了其全长序列，预测了4个编码蛋白的结构和功能。本项目的完成，不仅从转录水平上为不同猕猴桃品种对果腐病的抗性差异提出了一个较合理的解释，而且挖掘出一批猕猴桃果实熟腐病抗病相关基因，克隆了部分关键基因的全长序列并进行功能分析，这对阐明猕猴桃抗果腐病的分子机制，对指导猕猴桃品种抗性的遗传改良具有重要的理论意义和应用价值。