目录
摘要 I
关键词 I
Abstract II
引言
引言 1
1 材料与方法 3
1.1 实验材料 3
1.2 小麦多基因敲除载体构建 3
1.3 基本的分子克隆反应体系 4
1.3.1 PCR反应体系 4
1.3.2 Golden Gate反应体系 4
1.3.3 无缝克隆反应体系 5
1.4 小麦原生质体制备及转化 5
1.5 二代测序样品制备与分析 6
1.5.1 二代测序样品制备 6
1.5.2 二代测序数据分析 6
1.6其他常见分子生物学实验 6
2 结果与分析 6
2.1 编辑载体构建 6
2.2 三个STU系统的编辑效率 7
2.3 三个STU系统的编辑特性 8
3 讨论 11
3.1小麦多基因编辑体系的选择和优化 11
3.2 STU多基因编辑系统的扩展与应用 11
3.3小麦多基因编辑的应用前景 13
致谢 14
参考文献 15
附录 16
CRISPR/Cas9介导的小麦多基因编辑体系的建立
摘要
小麦(Triticum aestivum L., 2n = 42, AABBDD) 是世界上最重要的粮食作物之一，也是我国仅次于水稻的第二大农作物，其生产关系到世界和我国的粮食安全。由于小麦是六倍体植物，染色体背景复杂，基因组庞大，大量基因存在三个拷贝且功能冗余，使小麦基因功能的解析变得较为困难。在多倍体的小麦中建立多基因编辑体系有利于多拷贝基因功能的解析以及直接用于性状的设计改良。单转录元件(single transcriptional unit, STU)系统利用一个转录本同时转录多个sgRNA的策略，具有效率高，构建快，且便于遗传操作的特点。本研究通过小麦原生质体系统，分析和比较了基于CRISPR/Cas9的 STUCas9、STUtRNA和STUCsy4三种gRNA表达系统，并实现了在小麦原生质体 *51今日免费论文网|www.jxszl.com +Q: #351916072# 
中同时敲除五个基因。深度靶向测序结果表明，三种STU系统在小麦原生质体中都可以实现多基因编辑，其中STUCsy4系统在五个靶点处的平均效率最高，达到82.7%，STUtRNA系统为65.7%，而STUCas9系统只有5.2%。本研究对小麦多基因编辑具有重要的参考意义。
ESTABLISHMENT OF MULTIPLEX GENE EDITING SYSTEMS MEDIATED BY CRISPR / CAS9 IN WHEAT
ABSTRACT
Bread wheat is an allohexaploid species ( Triticum aestivum L., 2n = 42, AABBDD), and it is the major staple crop worldwide, which is highly important for food security both in China and many other countries. It has a large and complex genome with three similar but not identical copies of most of its genes. Its large genome, high ploidy and high content of repetitive DNA make it unusually recalcitrant to functional genome analysis. it is essential to establish multiplex gene editing systems for wheat functional genome analysis and precise designing breeding. Single transcription unit (STU) system is a type of multiplex gene editing systems with high efficiency, fast construction workflow and easy genetic manipulation, as they utilize single transcription unit to express multiple sgRNAs. However, the STU systems have not been thoroughly tested or compared in wheat. Here, we established three of the STU systems (STUCas9, STUtRNA and STUCsy4) based on CRISPR/Cas9 to knock out five endogenous genes simultaneously in wheat protoplasts. Deep sequencing showed that all three systems can perform multiplex gene editing. The average efficiency of STUCsy4 system was the highest (82.7%), STUtRNA system was slightly lower (65.7%) , and STUCas9 system was the lowest (5.2%). This study should be valuable for multiplex gene editing in wheat.

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