CRISPR/Cas9介导的茶树基因组定向编辑系统构建及茶树遗传转化体系改良

CRISPR/Cas9 technique (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) is a novel powerful approach for targeted genome editing, by which we could knock out, replace or site modify the object DNA in a very simple and easy way. Since its establishment, the CRISPR/Cas9 technique has been successfully applied in many eukaryotic organisms, including more than 10 plant species. However, it has not been available for genome editing of tea plant (Camellia sinensis (L.) O. Kuntze) due to the problems regarding to the design of sgRNA, CRISPR/Cas9 expression vectors and genetic transformation of tea plant. In order to solve the existing key problems in the application of CRISPR/Cas9 technique to Camellia sinensis, we are going to develop an efficient CRISPR/Cas9 system and a rapid genetic transformation system for Camellia sinensis. At first, we will establish a web tool for the design of sgRNA sequence according to the transcriptome data or genome data (if available) from Camellia sinensis. Then a series of vectors, including intermediate vectors and destination vectors, will be constructed for the expression of sgRNA and Cas9 protein in Camellia sinensis cells. After that, the lines of Camellia sinensis somatic embryos, which are sensitive to Agrobacterium tumefaciens and are easy to form adventitious bud, will be selected and be multiplied to establish an excellent somatic embryo bank of Camellia sinensis. Finally, the transformation condition will be optimized by using response surface methodology. The present project would realize the application of CRISPR/Cas9 technique for genomic editing in tea plant, which would significantly improve the progress on breeding, functional characterization of novel genes and genetic engineering of tea plant. In addition, this project would provide a reference method for genome modification of other non-model plants.

CRISPR/Cas9 技术是一门新兴的基因组定向编辑技术，可轻松实现对目标基因的敲除、替换和定点突变等操作。该技术自诞生到现在，仅约三年的时间已迅速在多种动、植物中得以应用，其中植物就已达10余种。然而，CRISPR/Cas9技术在茶树基因组编辑中的应用，面临靶点设计、表达载体的有效性以及茶树的遗传转化等瓶颈问题。本项目旨在解决制约CRISPR/Cas9技术在茶树中应用的核心难题，拟充分利用现有资源，开发适用于茶树的sgRNA序列设计软件，构建能在茶树细胞中稳定表达的CRISPR/Cas9表达载体，同时筛选再生能力强、对农杆菌敏感的遗传转化受体，并确定最佳遗传转化条件，有望实现CRISPR/Cas9技术在茶树中的成功应用。该项目的实施将有力推动茶树分子育种、基因功能研究、茶树基因工程等工作实现质的飞越，同时也为其它非模式作物的基因组编辑提供新的思路，具有重要的学术价值和应用价值。

CRISPR/Cas9 技术是一门新兴的基因组定向编辑技术，可轻松实现对目标基因的敲除、替换和定点突变等操作。该技术自诞生到现在，迅速在多种动、植物中得以应用，其中植物就已达20余种。然而，CRISPR/Cas9技术在茶树基因组编辑中的应用，面临靶点设计、表达载体的有效性以及茶树的遗传转化等瓶颈问题。为解决制约CRISPR/Cas9技术在茶树中应用的核心难题，本项目对靶点设计软件、载体构建方法以及转化因素进行了研究。建立了适用于茶树基因组编辑的sgRNA序列设计平台，构建了能在茶树细胞中稳定表达的CRISPR/Cas9载体，同时筛选了再生能力强、对农杆菌敏感的遗传转化受体，优化了茶树遗传转化条件，建立了茶树体细胞胚与再分发的最佳培养方法，实现了CRISPR /Cas9技术在茶树中的成功应用。通过对已有茶树基因组数据整合与整理，利用CRISPR primer designer构建了茶树基因组编辑数据库，该困适用于茶树基因组sgRNA序列设计；利用pYLCRISPR/Cas9系列载体成功对茶树目标基因进行了靶点突变，获得了稳定表达的转基因体胚。此外，探讨了阻碍茶树遗传转化的分子机制，通过转录组学的方法分析了农杆菌在转化茶树叶片与烟草时基因表达的差异，结果表明在转化茶树过程中多个基因的表达受到影响，如VirE2基因表达量远低于转化烟草时的表达量，该结果为进一步提高茶树的转化效率奠定了基础。最后，采用本项目建立的技术平台，验证了多个茶树新基因的功能，证实了其实用性。该项目为茶树分子育种、基因功能研究、茶树基因工程等研究的顺利开展提供了新的工具，同时也为其它非模式作物尤其是自交不亲和的木本作物的基因组编辑提供了新的思路。