利用CRISPR/Cas9 技术研究FvNBS家族基因在草莓炭疽病抗性调控中的功能

Strawberry anthracnose caused by C. gloeosporioides have led to serious losses in field production. However, the functional analysis of resistance gene to strawberry anthracnose is still scarce. Here, by RNA-Seq, we have screened out some different expressed FvNBS family genes in anthracnose susceptible strawberry cultivar (JiuXiang) at different stages. Through optimizing CRISPR/Cas9-mediated genome editing in strawberry and genetic transformation, we observe resistance of strawberry anthracnose in the FvNBSs sequence deleted mutants after infected with C. gloeosporioides. Moreover, We further analyze the disease-resistant mechanism of FvNBSs to anthracnose in strawberry by subcellular localization and trypan blue staining. Finally, we can obtain some of FvNBSs genes which have an importent function in resistance to strawberry anthracnose. What's more, on the one hand the project can provide an effective CRISPR/Cas9-mediated gene editing technique in strawberry which can be successfully used in the research of gene function; on the other hand functional analysis of FvNBSs gene in resistance to strawberry anthracnose can provide a very powerful theoretical basis for breeding of excellent disease-resistant germplasm in the future.

草莓炭疽病是为害草莓生产的主要真菌病害之一，然而有关草莓炭疽病抗病基因功能研究目前还很少。NBS类抗病基因被广泛认为在效应子触发的诱导性免疫中起着非常重要的作用。本项目通过转录组测序发现FvNBSs基因在我国优势草莓炭疽菌C. gloeosporioides浸染高感‘久香’幼苗后不同阶段有差异性表达现象，拟采用草莓CRISPR/Cas9基因编辑技术和转基因技术，观察分析FvNBSs基因缺失突变后对炭疽病抗性调控功能。同时，我们还通过亚细胞定位和台盼蓝染色等手段进一步观察分析FvNBSs基因的抗病机制。本项目优化建立草莓CRISPR/Cas9基因编辑技术体系，初探FvNBSs基因在草莓炭疽病抗性调控中的功能，创新了草莓基因功能研究的新技术，并为未来培育优质抗炭疽病草莓新种质选育提供了有力的理论依据。

草莓炭疽病是危害草莓生产的主要真菌病害之一，然而有关草莓炭疽病抗病基因功能研究目前还很少。本项目主要集中探索NBS类抗病基因在草莓炭疽病抗病性方面的功能。项目研究结果表示：（1）本项目通过转录组测序分析在栽培品种‘久香’中分类、鉴定了232个FaNBS抗病编码基因，并对FaNBS-ID类抗病进行了保守结构域分析；（2）转录组数据结合QT-PCR分析发现了4个关键FvNBSs抗病基因（mrna24117.1-v1.0-hybrid，mrna05887.1-v1.0-hybrid，mrna35167.1-v1.0-hybrid和mrna00463.1-v1.0-hybrid）表达量和炭疽菌C. fructicola浸染有一定相关性；（3）本项目尝试利用3种不同的植物基因编辑系统构了关键抗病NBS基因载体共52个编辑载体，通过测序验证并成功转化到农杆菌GV3101菌株中；（4）本项目已经成功建立了草莓野生种植‘夏威夷4号’、‘YW’和栽培品种‘红颜’和‘申琪’的草莓转化体系；（5）通过GFP荧光信号检测和CAS9基因检测共获得了25个稳定遗传转化的T0代阳性转基因株系；（6）VIGS实验验证FvNBS15887和FvNBS00463抗病基因敲除之后可以促进草莓炭疽病发病进程；（7）本项目已经成功克隆了mrna24117.1-v1.0-hybrid，mrna05887.1-v1.0-hybrid，mrna35167.1-v1.0-hybrid和mrna00463.1-v1.0-hybrid 4个关键抗病NBS基因的CD全长序列；（8）初步亚细胞定位结果显示FvNBS00463抗病基因定位在细胞质和细胞膜中；（9）通过对不同品种做接菌实验分析发现，‘申阳’是中抗品种，‘久香’是中感品种，同时外施水杨酸可以缓解炭疽菌在草莓叶片上的发病进程；（10）台盼蓝染色实验发现，炭疽菌在草莓植物体内接种后12 hpi分生孢子开始萌发，到24 hpi时从两个尖端产生了一根胚芽管， 72 hp时菌丝的形成具次级菌丝的形态，到96 hpi，次级菌丝占主导地位，同时有大量的叶片附着胞；（11）通过酵母筛库我们筛选到了草莓BAG1基因和NBS抗病基因NBS35167互作，进一步通过酵母双杂交实验验证了互作。.以上本项目研究结果为草莓炭疽病抗病机理研究奠定基础，同时为未来培育优质抗炭疽病草莓新种质选育