摘要：

为了探明葡萄抗霜霉病基因的抗病机制,分别以感病品种玫瑰香和抗病品种摩尔多瓦叶片为试材,离体接种葡萄霜霉病菌,在光照培养箱中培养。用JEOL-6360型扫描电子显微镜观察霜霉菌侵染不同品种叶片后的发育情况,并利用MGISEQ-2000高通量测序平台对2个品种进行转录组测序及差异表达基因分析。结果表明,玫瑰香在接种4 d后叶片背面出现肉眼可见的白色霉层,病斑随时间延长面积增大,叶片正面相应位置出现褪绿黄斑,而在摩尔多瓦叶片上仅在接种点处有少量黑斑。电镜观察结果显示,玫瑰香叶片上病原菌孢子侵入气孔,萌发长出新的孢子囊梗,释放出孢子,在叶片上繁殖;摩尔多瓦叶片上的孢子囊数量少,大部分气孔关闭,气孔周围有少量孢子、菌丝和组织碎片分布。转录组数据共获得约664.86 Mb高质量序列,与玫瑰香相比,在摩尔多瓦中检测到5 095个差异表达基因,其中2 975个为上调表达基因,2 120个为下调表达基因。GO分析显示,差异表达基因主要富集在生物学途径(代谢过程、细胞过程、对刺激的反应和生物调节类别)、细胞组分(细胞、膜、膜组分和细胞器)和分子功能(催化活性和组装、转运体活性、转录调节因子活性)三大类。KEGG分析差异表达基因,主要富集在植物病原菌互作途径和MAPK信号转导途径等通路中。选取其中9个可能与葡萄抗病性相关的基因,qRT-PCR结果表明,9个候选基因在葡萄响应霜霉菌侵染过程中发挥一定的作用。

关键词: 葡萄霜霉病, 抗病基因, 电镜, 转录组测序

Abstract:

In order to explore the mechanism of grape defense response to grape downy mildew,the leaves of Muscat and Moldova were inoculated with grape downy mildew in vitro and cultured in light incubator.The development of downy mildews was observed from macroscopic and microscopic aspect.In macroscopic,it was showed that white mildew layer appeared on the back of leaves of Muscat four days after inoculation,and the area of disease spots expanded with time,meanwhile chlorosis yellow spots appeared on the opposite position of the front side of leaves.However,there were only a few black spots on leaves of Moldova at the inoculation point.Under electron microscope,it was found that the spores of the pathogen invaded the stomata and colonized,and germinated new sporangium stalks,which released the spores and propagated on the leaves of Muscat.But the number of sporangia was small on the leaves of Moldova,and most of the stomata was closed,only with spores,hyphae and tissue fragments scattered around the stomata.Furthermore,transcriptome sequencing and differentially expressed genes were analyzed by Illumina high-throughput sequencing.A total of 664.86 Mb of high-quality reads were obtained by RNA-seq.GO enrichment analysis showed that differentially expressed genes were mainly concentrated in biological pathways(metabolic process,cellular process,response to stimulus and biological regulation category),cell components(cell, membrane, membrane part and organelle),molecular functions(catalytic activity and binding,transporter activity and transcription regulator activity).KEGG enrichment analysis showed that the differentially expressed genes were mainly enriched in the plant pathogen interaction pathway and MAPK signal transduction pathway.Nine genes that may be related to grape disease resistance were screened and verified by Real time PCR analysis,and the results were consistent with the transcriptome sequencing results.

Key words: Downy mildew, Disease resistance gene, Electron microscopy(SEM), Transcriptome sequencing(RNA-seq)