靶向核酸酶技术体系在小麦基因组定位修饰中的建立及应用研究

Engineered nucleases, such as Zinc Finger nuclease (ZFN) and Transcription Activator-Like Effector Nuclease (TALEN), have been recently utilized to create high efficient targeted genome modification in model plants and animals. Such nucleases can be designed to recognize and induce double-strand DNA breaks at specific genomic loci. These breaks are subjected to cellular DNA repair processed that often result in sequence alteration at target loci with remarkably high frequencies. Although nuclease-mediated genome modification has been demonstrated in several plant species, including Arabidopsis, tobacco and maize, it has been indicated that the technology platform has to be customized for each target species based on its genome complexity, polyploidy and suitable transformation tools. In this proposal, we aim to first establish a high efficient ZFN and TALEN platform for precise genome modification in wheat. ZFNs and TALENs with high efficacy and sequence specificity will be designed, evaluated and tested taking into account of complexity and polyploidy of wheat genome. Customized ZFNs or TALENs will be made to target exogenous selection or marker genes and endogenous secalin gene. Specific aims include: 1) to demonstrate highly efficient gene disruption in pre-integrated selection marker genes; 2) to generate mutant lines with disrupted secalin gene or gene family; 3) to demonstrate and optimize targeted gene replacement at the secalin locus. Modified wheat lines will be further followed through traditional breeding for phenotype test in progenies. Completion of the proposed research will establish a new paradigm in wheat genetic manipulation for basic and applied research.

通过引入靶向核酸酶（ZFN、TALEN）造成目标位点DNA双链断裂，在细胞内源修复系统作用下，可实现目标基因组精确定位修饰，是下一代生物遗传修饰技术的关键所在。本研究以靶向核酸酶技术体系在小麦中的构建为出发点，针对小麦基因组结构特性及ZFN、TALEN设计原理，着力搭建高效率、高活性、高特异性的小麦靶向核酸酶技术平台，重点提高ZFN、TALEN对小麦多倍体基因组同源序列的识别特异性及构建高效率筛选方案。在此基础上，针对小麦基因工程常用选择及标记基因、小麦1RS-1BL易位系黑麦碱基因，构建及筛选高效ZFN、TALEN，实现：（1）选择及标记基因的高效敲除；（2）黑麦碱基因的特异性敲除或基因簇缺失；（3）黑麦碱位点的BAR基因重组置换。进而结合常规育种途径，分离、筛选、固定各类型基因组修饰后代株系，为合理、有效应用靶向核酸酶进行小麦基因功能分析及种质创新提供理论依据和物质基础。

依据项目计划书陈述内容，课题组以序列特异性核酸酶（sequence-specific nuclease, SSN）技术体系在小麦中的构建为出发点，针对小麦基因组结构特性及SSN工作、 设计原理，着力搭建高效率、高活性、高特异性的小麦基因组定向修饰技术平台，实现基于SSN介导的高效目标基因组定向修饰。项目实施四年以来，按照计划书内容，针对“小麦基因组定向修饰技术平台构建及应用”核心研究目标开展工作，主要成果如下：1）完成了小麦TALEN骨架载体优化、“双RVD单元”高效TALEN组装文库构建，实现了小麦目标基因有效定向剪切；2）开发了基于单链构象多态性（SSCP）的植物基因组定向修饰快速基因分型体系；3）构建了小麦选择、标记及内源基因活性SSN定向修饰及重组载体；4）构建了基于Golden Gate反应的高效植物CRISPR-Cas9骨架载体文库；5）开发了高效CRISPR-Cas9单一转录单元（single transcript unit CRISPR-Cas9, STU-Cas9）定向修饰系统；6）完成了基于CRISPR-Cpf1新系统的植物基因组定向修饰；7）以小麦、水稻模式材料，针对重要农艺性状及microRNA编码基因，有效创制了定向修饰育种材料，初步完成了农艺性状及稳定性分析。基于以上研究成果，截止目前本项目共发表研究论文13篇（其中SCI论文12篇、ESI高被引及热点论文1篇）、受邀学术报告2次、申请发明专利3项。本项目的实施，提升了植物基因组定向修饰效率，为合理、有效应用基因组定向修饰技术进行小麦、水稻等作物基因功能分析及种质创新提供了技术平台及材料基础。