利用CRISPR技术敲除蒺藜苜蓿RIC8基因的初步研究
毕业论文关键词：蒺藜苜蓿；RIC8；根毛变形；结瘤因子；crispr
A preliminary study on the application of CRISPR techniques to knock out RIC8 gene of Medicago truncatula
Abstract：As a model plant in legume, Medicago truncatula is an ideal material for studying the regulation mechanism of interaction between legume and Rhizobium symbiotic. Rhizobia preferentially enter legume root hairs via infection threads, after which root hairs undergo tip swelling, branching, and curling. The RIC family is an effector of small G protein ROP, involving in ROP signaling on the cell polar growth. ROP functions on regulating cell polarity, participate in plant microbe interaction, and hormone signal transduction. Medicago truncatula ROP10 was reported to involve in the process of nod factor-induced root hair deformation. This study will construct one member of RIC family (RIC8) knockout vector by crispr technology. Then the vector will transform into Medicago truncatula to provide a foundation for studying the nodulation factor in induced root hair deformation in the process of function.
Key words: Medicago truncatula；RIC8；root hair deformation；nod factor；crispr
目 录
摘要1
关键词1
Abstract…1
Key words1
1 引言2
1.1 蒺藜苜蓿遗传转化的研究进展 2
1.2 根毛参与豆科植物与根瘤菌的共生固氮过程…2
1.2.1 根毛的生长模式…2
1.2.2 根毛的变形过程…3
1.3 结瘤因子…3
1.4 ROPs和RICs的功能介绍3
1.5 CRISPR/Cas系统的功能介绍…4
1.6 本论文研究目的和意义4
2 材料与方法…5
2.1 实验材料…5
2.1.1 植物材料…5
2.1.2 菌株和质粒载体5
2.1.3 仪器设备…5
2.2 试剂配制6
2.2.1 常用试剂和溶液的配制…6
2.2.2 常用培养基的配置…6
2.3 实验方法…7
2.3.1 构建CRISPR/Cas9-RIC8打靶载体7
2.3.2 蒺藜苜蓿转化（农杆菌介导法）…9
2.3.3 CTAB法提取蒺藜苜蓿DNA…9
3 结果与分析10
3.1 构建CRISPR/Cas9-RIC8打靶载体10
3.2 CRISPR/Cas9-RIC8蒺藜苜蓿植株的获得10
3.2.1 共培养10
3.2.2 培养愈伤组织…11
3.2.3 胚的形成…12
3.2.4 生根…12
3.2.5 温室培养13
3.3 对转基因蒺藜苜蓿的分子检测13
4 讨论与展望13
4.1 CRISPR/Cas9技术的应用意义13
4.2 蒺藜苜蓿遗传转化的研究意义14
致谢…14
参考文献15
利用CRISPR技术敲除蒺藜苜蓿RIC8基因的初步研究
1 引言
1．1 蒺藜苜蓿遗传转化的研究进展
蒺藜苜蓿（Medicago truncatula）是豆科苜蓿属的一年生植物，也被称为截形苜蓿或截叶苜蓿。蒺藜苜蓿的优势体现在：生长周期短，只需要80至100天就可以成熟；染色体组小，2n=16便于观察；基因组小，便于分析和修饰；遗传转化效率高，降低实验难度；自花授粉，不需要担心配种问题；共生固氮等[1]。蒺藜苜蓿与紫花苜蓿等豆科植物有亲缘关系，基因组间具有一定程度上的同源性，所以借助蒺藜苜蓿的实验成果及其基因资源对促进以紫花苜蓿为代表的其它豆科作物的遗传修饰和改造显得十分重要。