利用dCas9基因激活技术高通量筛选水稻抗病基因的探索

Pathogen infection has made severe damages to the yield of major crop rice, however, the available defense gene resources in rice is insufficient. Therefore, it’s essential to comprehensively exploit the defense gene resources in rice for breeding new pathogen-resistant rice varieties. As our primary results showed that the rice genes could be activated by sgRNA mediated dCas9-TV, we propose to construct a high-throughput strategy for gene gain-of-function screening through an integrated utilization of dCas9-TV transcriptional activator, rice protoplast system, flow cytometry sorting, and high-throughput sequencing. Firstly, we will construct transgenic rice contained dCas9-TV and immunity report system. Secondly, a sgRNA library targeting to the promoters of all the rice genes will be established and transfected the protoplasts of the transgenic rice. Thirdly, GFP-activated cells will be sorted by flow cytometry and the relevant sgRNAs will be amplified by PCR for high-throughput sequencing. Fourthly, sgRNAs significantly associated to immunity activation will be identified using bioinformatics analysis. Finally, a batch of candidate genes positively regulating rice immunity will be identified. Comparing to the traditional overexpression transgene strategy, this more efficient and time-saving strategy can circumvent the technical problems including transgenic plant growth suppression caused by continuous immunity activation and gene loss-of-function caused by T-DNA insertion. Hence, this high-throughput screening strategy is a potential approach for identifying novel defense genes in rice.

病害严重影响主要粮食作物水稻的产量，但目前可利用的水稻抗病基因资源相对缺乏，故挖掘抗病基因资源对培育抗病新品种具有重要意义。在前期证实dCas9-TV在sgRNA介导下激活水稻基因的基础上，本项目拟结合dCas9-TV转录激活因子、水稻原生质体系统、流式细胞分选和高通量测序建立一种高通量基因gain of function筛选策略。首先构建dCas9-TV/免疫报告系统转基因水稻; 然后用靶向水稻所有基因启动子的sgRNA文库转染该转基因水稻原生质体；接着流式分选GFP显著激活的细胞，PCR扩增sgRNA片段进行高通量测序；再用生物信息学分析找出与免疫激活显著关联的sgRNA；最后鉴定一批正向调控水稻免疫的抗病基因。相比传统的转基因过表达策略，该策略能避免免疫持续激活拮抗植物生长和T-DNA插入导致基因功能缺失等技术难题，且更高效省时。因此该策略有望成为高通量筛选新水稻抗病基因的有力手段。

病害严重危害粮食安全，培育高抗病品种是一种重要的解决方法，但目前可利用的水稻抗病基因资源相对匮乏。我们前期证实dCas9-TV在sgRNA介导下可以激活水稻基因，而且原生质体系统有助于进行高通量筛选。在本项目的资助下，我们进行了利用dCas9-TV转录激活技术高通量筛选水稻抗病基因的探索，并获得以下主要发现：利用真菌几丁质或稻瘟菌处理的水稻愈伤组织进行转录组学分析和RT-qPCR验证获得了一批新水稻免疫响应基因；设计和构建了靶向水稻OsRLK和OsRLCK基因家族启动子的sgRNA文库；筛选并证实OsChIT6pro::INTACT可作为水稻免疫响应报告系统；使用INTACT方法能富集免疫激活的细胞和鉴定潜在抗病基因；对高通量筛选获得的抗病基因进行了初步的功能验证。由此，我们建立了一套dCas9-TV转录激活技术高通量筛选水稻抗病基因的策略，并为培育抗病水稻新品种提供了新基因资源。我们同时还研究揭示了细胞质类受体激酶PBL19所介导的一条新型几丁质信号转导通路，为植物抗真菌机制提供了新的认知。本研究所取得的主要成果正在整理成文，其余成果已发表一篇Nature Plants论文。