核桃抗胶孢炭疽菌的先天免疫系统转录组和蛋白质组解析

Walnut (Juglans regia L.) has 2n=32 chromosomes and approximately 606 Mb per 1C genome. It is cultivated for its nuts and timber, and serve as prominent ecological niche species in hardwood forests. Walnut anthracnose induced by C. gloeosporioides had occurred and caused seriously damage for walnut trees. In this project, Lifestyle transitions in GFP-m9 strain and the changs of walnut infected tissue were observed. The uninfected tissues, attachment, biotrophy, necrotrophy and 15dpi uninoculated distal tissues were employed as test sample. About 50000 unigenes per sample were expected to be obtained by Roche 454 GS FLX sequencing. The unigenes annotation and expression levels were analysised to identify the different expression unigenes in five type tissues. Protein identification and quantification was carred out with iTRAQ-nano-LC-MS/MS analysis and about 700 proteins were expected to be quantified during above five stages of walnut tissues. Proteomic data analysis and functional interpretation was performed to distinguish about 300～400 proteins with differential expression. The pattern of correlation between the differential expression unigenes and the different expression proteins was estimated to explore key protein/transcript at PTI、ETI and SAR pathway in walnut. This project will contribute to better understanding of innate immunity system conferring resistance to C. gloeosporioides and facilitate novel resistance genes discovery, marker-assisted selection that would accelerate breeding progress of walnut.

核桃是重要的"木本粮油"生态树种，2n=32，全基因组约606Mb。由C.gloeosporioides引起的核桃炭疽病危害严重。本项目观察炭疽菌菌株GFP-m9侵染核桃抗病品种'瑞嘉'果实青皮的过程，解剖学研究被侵染组织形态学变化；以未侵染组织，侵染初期、营养寄生期、腐生寄生期的病部组织和接种后15d未接种的远端组织为样本；Roche 454 GS FLX测序，每样本获得约50000条以上可功能注释的Unigene，对Unigene功能注释和表达量分析，筛选差异表达基因；利用iTRAQ-nano-LC-MS/MS技术，获得700个以上可定量蛋白，确定在不同组织差异表达蛋白300～400个，分析其在代谢通路的功能；分析表达谱和蛋白质组的相关性，发掘核桃中参与PTI、ETI和SAR过程的关键基因/蛋白质。研究将解析核桃抗炭疽病的先天免疫系统，为核桃分子设计育种奠基础。

核桃（Juglans regia L.）是重要的木本油料树种，但由胶孢炭疽菌Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. 引起的核桃炭疽病（anthracnose）对我国核桃产业的危害日趋严重。为了在转录组和蛋白质组水平探讨核桃抗炭疽病的特点，申请开展了本项目的研究。项目实施以来，开展的主要工作如下：.1，以核桃抗炭疽病单株 B26 的果实（F26）、感病单株 B37 的叶片（L37）、叶片抗病而果实易感病单株 4-23 的叶片（L423）和果实（F423），为 4 份供试植物材料；以 C. gloeosporioides 菌株 m9 为供试菌株。.2，形态学和解剖学观察了 C. gloeosporioides 对核桃叶片和果实的侵染过程。确定接种 0 h（未侵染），接种后 24 hpi（附着孢形成）、48 hpi（初生菌丝产生）、72 hpi（次生菌丝形成）感病组织和 120 hpi 未感病组织为取样部位。.3，测定了供试样本的几丁质酶（Chitinase）、β-1,3葡聚糖酶（β-1,3-GA）、H2O2、过氧化氢酶（Catalase，CAT）、NO、和 Ca+ 的变化动态，探讨了在C. gloeosporioides 侵染过程中核桃组织的生理响应。.4，对 4 份供植物材料的5个时间节点，共20份样本（每样本重复2次）用 Illumina Hiseq4000 测定了其转录组数据；同时，用 Thermo Q-Exactive 型质谱仪对其进行 Label-free的质谱分析。转录组分析获得 62683 个已知的转录本与 178630 个未知转录本，38541 个已知的基因与 124017 个未知的基因；蛋白质组分析共鉴定出 29798 个肽段和 4932 个蛋白质。.5，利用差异基因、蛋白质表达分析，时间序列基因、蛋白质表达分析，加权基因、蛋白质共表达分析和基因-蛋白质关联分析，以及 C. gloeosporioides与核桃的互作等策略，分析了核桃叶片和果实响应 C. gloeosporioides 侵染过程不同阶段上的主要基因、蛋白质及其功能与代谢通路。.6，项目实施期间，发表SCI论文2篇，7 名研究生的毕业论文得到了项目资助，获得奖励 3 项。参加的重要学术会议 7 次。