利用CRISPR/Cas9技术定点突变拟南芥CRWN3基因
Site directed mutagenesis of Arabidopsis CRWN3 gene by CRISPR/Cas9 technique
Abstract：CRISPR/Cas9 system is an immune mechanism , which widely existed in bacteria and archaea and can effectively help the bacteria to resist a series of exogenous DNA infection including phage and plasmid. Compared with the traditional gene editing technology, its mediated genomic editing technology has the advantages of simple operation, high efficiency, wide application range and low cost. In this paper, we use Arabidopsis thaliana wild type (Columbia ecotype Col) as material,through the CRISPR/Cas9 system, use snapgene to design a single sg RNA; this single sg RNA is used to construct targeted vectorn and mutate single gene CRWN3 target site; After infecting the Arabidopsis thaliana Col-0 plants, we screened the mutant out and purified the mutant positive plants. Finally, we obtained the T2 generation heterozygous mutants of the target site of CRWN3 gene of Arabidopsis thaliana.
Key words: CRISPR/Cas9;Single genomics editing; Arabidopsis;CRWN3 gene.
目 录
摘要： 1
关键词： 1
Abstract： . 1
Key words: 1
引言 1
1 材料与方法 2
1．1 实验材料 2
1．1．1 拟南芥材料 2
1．1．2 实验菌株 2
1．1．3 克隆载体 2
1．1．4 实验仪器设备 2
1．1．5 实验培养基和试剂的配制 3
1．2 实验方法 3
1．2．1 植物材料的培养 3
1．2．2 设计sg RNA 3
1．2．3 构建载体 4
1．2．4 侵染拟南芥植株 6
1．2．5 突变阳性植株筛选 7
2 结果与分析 8
2．1 符合要求 sg RNA 序列 . 8
2．2 构建敲除 CRWN3 的CRISPR 载体 . 8
2．2．1 sg RNA连接到 AtU6载体的大肠杆菌菌液 PCR 8
2．2．2 连接产物转化大肠杆菌 DH5α提取质粒后测序结果 8
2．2．3 目的片段转入农杆菌菌液 PCR 9
2．3 潮霉素抗性板子上的 T0代阳性苗 . 9
2．4 T0代阳性苗酶切验证 10
2．5 成功敲除基因的个体单克隆测序结果 11
2．6 T1代阳性苗酶切验证 11
3 讨论 12
致谢 12
参考文献： 13
附录 14
毕业论文引言
随着二十世纪末人类基因组计划的启动和完成，多种多样植物的基因组计划也相继启动并发展迅猛[1]，特别是以拟南芥、水稻等为代表的模式植物基因组研究已取得了卓有成效的发展[2]