植物基因组和碱基编辑工具箱的创制及功能验证

发布时间：2018-03-27 06:41

本文选题：CRISPR/Cas9　切入点：双元载体　出处：《中国农业大学》2017年博士论文

【摘要】：对于生命规律的探究很大程度上依赖于基因功能的阐释,而全基因组测序及基因组学的发展使得对强大的基因组编辑工具的需求更为迫切。CRISPR/Cas9基因组编辑技术以其设计原理简单、技术门槛低、操作便捷、通用性强等特点在2013年初迅速成为基因组编辑研究领域的关注焦点。为了测试CRISPR/Cas9在植物中的应用效果,建立全面的CRISPR/Cas9工具系统,助力植物基因功能的研究,加快对植物功能基因的挖掘,本研究工作首先基于CRISPR/Cas9技术创制了植物的基因组编辑工具箱,涵盖各种双元载体和sgRNA模板载体以及相应的sgRNA组装方法。工具箱包括适用于双子叶植物的双元载体pHSE401、pBSE401、pKSE401和适用于单子叶植物的双元载体pHUE411、pBUE411。通过在玉米和拟南芥中对CRISPR/Cas9工具箱进行功能验证,证明该CRISPR/Cas9工具箱在双子叶和单子叶植物中均能高效诱导基因组编辑,因此可为相关研究人员提供更为简单、便捷的工具平台,实现高效的突变体创制目的。在应用CRISPR/Cas9工具时,我们发现以胚性愈伤为转化受体的单子叶植物玉米可以在T0代高效产生纯合或双等位突变体,但转化方法最简单高效的模式植物拟南芥的T1代突变体却存在严重的嵌合现象,影响了突变传递到下一代的效率。我们猜测转基因T1代嵌合体的形成可能是与使用的CaMV 35S等组成型启动子在卵细胞或者单细胞期胚中的活性较低有关。为此,本研究采用卵细胞特异性启动子驱动Cas9,创制了卵细胞特异性表达的CRISPR/Cas9系统,即EPC(egg cell-specific promoter-controlled)CRISPR/Cas9 系统,接着对EPC CRISPR/Cas9 进行了功能测试,结果表明使用该系统在拟南芥的转基因T1代成功地实现了两基因(CHLI1 CHLI2)和三基因(TRY CPC ETC2)的纯合突变,解决了CRISPR/Cas9技术在拟南芥中应用时遇到的T1代突变体嵌合严重的问题。上述三基因和两基因同时发生纯合或双等位突变的的株系占相应的全部T1代转基因植株的比例可以分别达到8.3%和13.1%。通过对三种启动子(EC1.2p/35Sp/EC1.1p)和两种终止子(E9t/Nost)进行组合,比较相应载体的打靶效率发现,Cas9终止子的选择对于EPC CRISPR/Cas9发挥作用至关重要。此外,为了进一步提高EPCCRISPR/Cas9系统的基因打靶效率,采用启动子融合策略对EPC CRISPR/Cas9系统进行了优化尝试。通过尝试不同的增强子与EC1.2或Ec1.1的启动子进行融合,发现将EC1.2的增强子与EC1.1的启动子融合后显著提高了EPC CRISPR/Cas9系统对靶基因的纯合突变效率(17.0%),达到了优化的目的。CRISPR/Cas9和胞嘧啶脱氨酶介导的碱基编辑系统,最先在动物中得到成功应用。为了测试碱基编辑系统在植物中的应用效果,本研究创制了一个适用于双子叶植物(pHSE901、pBSE901、pKSE901、pHEE901)和单子叶植物(pHUE911、pBUE911)的碱基编辑工具箱并在拟南芥中进行了测试。通过对T1代和T2代靶基因AtALS的编辑效果分析发现,在T1代得到靶基因编辑的株系占全部株系的1.7%(4/240);在T1代得到编辑的靶点可以高效地传递到T2代,其中三个T1株系的T2代植株中,获得除草剂抗性的碱基编辑植株的平均比例达84.1%。此外,研究结果提示在植物中,PAM区可能是一个新型的"脱氨酶作用窗口"。总之,本研究中建立的植物基因组编辑和碱基编辑系统无论在基础理论研究还是作物改良中都有很好的应用前景。
[Abstract]:For exploring the law of life depends largely on the interpretation of gene function, and the development of whole genome sequencing and genomics makes powerful genome editing tools is more urgent than.CRISPR/Cas9 genome editing technology with its simple design principle, low technical threshold, convenient operation, strong versatility and so quickly become the focus of research field genome editing in early 2013. In order to apply the test results of CRISPR/Cas9 in plant, the establishment of CRISPR/Cas9 tool system comprehensively, research on power plant gene function, accelerate the mining of plant gene function, this paper firstly based on the CRISPR/Cas9 technology to create a plant genome editing toolbox, covering a variety of binary vector and sgRNA vector template and the corresponding sgRNA assembly method. Toolbox includes binary vector pHSE401 for dicotyledonous plants, pBSE401, PKSE401 and for monocotyledon binary vector pHUE411, pBUE411. through the functional verification of the CRISPR/Cas9 toolbox in maize and Arabidopsis, prove that the CRISPR/Cas9 toolbox can effectively induce genome editing in dicotyledonous and monocotyledonous plants, so it can be for the relevant researchers to provide more simple, convenient tool platform, implementation efficient creation. Mutants in the application of CRISPR/Cas9 tools, we found that the embryogenic callus as transformation receptor monocots maize can be efficiently generated homozygous or double mutant allele in T0 generation, but the transformation of the most simple and efficient method of Arabidopsis T1 mutant generation has seriously affected the chimeric, mutation the efficiency of transfer to the next generation. We hypothesized that the transgenic T1 generation of chimera formation may be associated with the use of the CaMV 35S constitutive promoter in oocytes or Single cell embryos in lower activity. Therefore, this study uses the egg cell specific promoter Cas9, the creation of the egg cell specific expression of CRISPR/Cas9 system, namely EPC (egg cell-specific promoter-controlled) CRISPR/Cas9 system, and then tested the EPC CRISPR/Cas9, the result shows that the system of T1 in transgenic Arabidopsis thaliana the generation successfully realized two gene (CHLI1 CHLI2) and three genes (TRY CPC ETC2) the homozygous mutant, T1 mutant chimeric generation solves the application of CRISPR/Cas9 technology in Arabidopsis have the serious problems. The three genes and two genes were homozygous or biallelic Mutation Strains accounted for all T1 corresponding transgenic plants at the same time the proportion can reach 8.3% and 13.1%. respectively through three kinds of promoter (EC1.2p/35Sp/EC1.1p) and two (E9t/Nost) of the terminator group, compared with corresponding Find the vector targeting efficiency, Cas9 terminator selection play a crucial role for the EPC CRISPR/Cas9. In addition, in order to further improve the gene targeting efficiency of EPCCRISPR/Cas9 system, using the promoter fusion strategy was optimized to try EPC CRISPR/Cas9 system. Through fusion to try different EC1.2 or Ec1.1 enhancer and promoter, enhancer and discovery the EC1.1 EC1.2 promoter fused significantly improved EPC CRISPR/Cas9 system on target gene homozygous mutation efficiency (17%), to achieve the purpose of.CRISPR/Cas9 and the optimization of cytosine deamination enzyme mediated DNA Editing System, the first is successfully applied in the animal. In order to test the application effect of base editing system in plants, this study created a suitable for dicotyledonous plants (pHSE901, pBSE901, pKSE901, pHEE901) and Monocotyledoneae (pHUE911, pBUE911) of the base Edit the toolbox and tested in Arabidopsis. Based on T1 and T2 generation AtALS target gene editing effects analysis showed that the obtained target gene editing strains accounted for 1.7% lines in T1 generation (4/240); get the target of editing can be efficiently transferred to T2 generation in T1 generation, the three strains of T1 plants in T2 generation, the average proportion of herbicide resistant plants obtained base edit up to 84.1%. in addition, the results of the study suggest that in plants, PAM may be a new type of "ADA window." in short, application prospect in this paper plant genome editing and editing system is both the base in the theoretical research and crop improvement are.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q943.2

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