渐狭叶烟草高效基因编辑体系的建立与优化

	渐狭叶烟草高效基因编辑体系的建立与优化
	梁永鑫
导师	吴劲松
关键词	渐狭叶烟草，基因编辑，农杆菌，CRISPR/Cas9 Nicotiana attenuata, Gene editing, Agrobacterium-mediated transformation, CRISPR/Cas9
摘要	渐狭叶烟草（Nicotiana attenuata)是一种野生二倍体的烟草，近年来已经发展成为烟草属植物中研究植物与昆虫、植物与病原菌互作的模式植物。目前，渐狭叶烟草中造成基因功能沉默的主要研究方法有两种，分别是病毒介导的基因沉默（virus induced gene silencing, VIGS）技术和基于农杆菌转化的RNA干扰（RNA interference, RNAi）技术，这两种方法均做不到基因功能的完全敲除。虽然在渐狭叶烟草中已经有基因编辑方法的报告，但是编辑效率不高。为此，我们提出以下的工程需求：建立并优化渐狭叶烟草的基因编辑体系，从而为以渐狭叶烟草为模式植物的基因功能研究提供更加快捷和高效的方法，为进一步深入研究其抗病和次生代谢调控奠定基础。因此，本论文以渐狭叶烟草为材料，分别通过病毒和农杆菌介导的方法，进行高效基因编辑体系的建立和优化，主要工程结果如下： 1）病毒介导的基因编辑方法的探索。利用成簇规律的间隔短回文重复相关蛋白-9核酸酶（Clustered regularly interspaced short palindromic repeats-associated protein-9 nuclease ，CRISPR/Cas9）系统是双元组分的特点，首先创制了含Cas9基因的转基因渐狭叶烟草，并对实验室的病毒载体pTV00进行改造，构建了融合报告基因八氢番茄红素脱氢酶基因(phytoene desaturase, PDS)的sgRNA及突变的FT的载体，通过农杆菌注射到表达Cas9基因的植物体内，以期实现病毒介导的基因编辑。但经测序发现并未出现预期的基因编辑，且在子代中也未观察到预期的白化表型。因此，利用病毒介导的基因编辑方法还需要进一步的摸索和探究。 2）利用pEB012载体的农杆菌介导基因编辑体系的探索。pEB012载体使用的是卡那霉素和红色(Production of Anthocyanin Pigment 1 , PAP1)筛选标记。利用pEB012载体，我们构建了PDS单靶点基因编辑载体。通过农杆菌浸染，愈伤诱导，抗性筛选，获得了一些PDS基因编辑的白化植株。通过测序分析，发现PDS基因编辑的类型主要为插入或者缺失突变。但实验中发现，野生型渐狭叶烟草对卡那霉素的本底抗性非常高，且只有在卡那霉素125 mg/L时有接近13 %的白化率。因此，我们认为该方法基因编辑效率不高且筛选较困难，仍需进一步的优化。 3）利用pHSE401载体，成功建立了基于农杆菌介导的高效基因编辑体系。我们利用潮霉素进行筛选的pHSE401双靶点基因编辑载体。构建了PDS基因双靶点基因编辑载体，转入农杆菌，通过浸染，愈伤诱导，潮霉素筛选，成功获得了接近60 %的PDS基因编辑效率的白化植株和白色愈伤。在这基础上，我们利用该体系，成功获得了多株WRKY70双靶点基因编辑的植物。并且WRKY70双靶点基因编辑的植物基因编辑的频率达到了83 %，远远超过了目前报道的30 %的编辑水平。因此，通过多种转化方式和多个载体的摸索和优化，我们最终建立了渐狭叶烟草目前基因编辑效率最高的体系——以潮霉素为筛选标记的基于pHSE401的双靶点基因编辑体系；并利用该体系成功获得了多株WRKY70双靶点基因编辑的植物，为下一步基因功能的研究奠定了扎实的基础。; Nicotiana attenuata, a wild diploid tobacco, has recently been developed as a model plant in the genus Nicotiana for the study of plant-insect and plant-pathogen interactions. Currently, there are two main methods to study the functional silencing of genes in N. attenuata. They are virus induced gene silencing (VIGS) and Agrobacterium transformation based RNA interference (RNAi) techniques. However, both methods do not create gene knock-out mutants, but rather generate plants with lower expression of target genes. Gene editing methods have been reported in N. attenuata, but the editing efficiency needs to be improved. Thus, establishing and optimizing the gene editing system of N. attenuata becomes an urgent engineering target for us. It will provide us an efficient method for gene function study in N. attenuata, and a foundation for further in-depth studies on genes involved in disease resistance and secondary metabolic regulation. Here, we tried to establish and optimize an efficient gene editing method in N. attenuata by using viral and agrobacterium as carriers. The main engineering results are as follows: 1) Exploration of virus-mediated gene editing methods. Taking the advantages of the feature that the CRISPR/Cas9 system is a binary component. We first created a transgenic N. attenuata plant with the Cas9 gene, and modified the laboratory viral vector pTV00 to construct a vector containing both the sgRNA of the reporter gene PDS and mutated FT. However, the expected gene editing events did not occur, and the expected bleaching phenotype was also not observed in the offsprings. Therefore, this method need to be further improved. 2) Exploration of Agrobacterium-mediated gene editing system by using pEB012 vector. The pEB012 vector contains a kanamycin and a red PAP1 screening marker. By using the pEB012 vector. We constructed a PDS single-target gene editing vector. Some calli were observed with bleaching phenotype after transformation. DNA Sequencing analysis revealed that the types of PDS gene editing were mainly insertion and deletion. However, the wild-type N. attenuata callus was highly resistance to kanamycin. They can grew on medium with kanamycin at 125 mg/L, in which conditions only around 13 % calli were bleaching. Therefore, further optimization is still needed. 3) A high efficient Agrobacterium-mediated gene editing system was successfully established by using the pHSE401 vector. This vector requires hygromycin for callus screening, rather than kanamycin. Two sgRNAs of PDS genes were constructed in the pHSE401 vector. Nearly 60 % of the calli were observed with bleaching phenotype, indicating high efficient of PDS gene-editing. By using this method, several WRKY70 dual-target gene editing plants were obtained. Moreover, the frequency of WRKY70 double-target gene editing in plants reached 83 %, far exceeding the current reported editing level of 30 %. Taken all together, we finally established the most efficient gene editing syste
语种	中文
	2022-05
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75106
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	梁永鑫. 渐狭叶烟草高效基因编辑体系的建立与优化[D]. 中国科学院大学,2022.

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