农杆菌介导的本生烟瞬时转化的影响因素探索
毕业论文关键字：本生烟；瞬时转化；细胞程序性死亡
Exploration of Influence Factors of Transient Agrobacterium-mediated Gene Expression System in Leaves of Nicotiana benthamiana
Abstract: Transient expression has become a widely used technology in biology research, mainly involved in gene functional analysis, subcellular localization, improvement and breeding of plant. In this study, we operated with Nicotiana Benthamiana as experimental materials，AGL1,GV3101 and LBA4404 three common agrobacterium strains, using self-activated mutant of barley disease-resistant protein MLA, MLA10(D502V), as reporter gene, diluted the bacteria into different OD value and then infect tobacco leaves by transient transformation and observe the degree of programmed cell death to find the optimized condition of transient expression in Nicotiana Benthamiana. According to this study, we found that injecting Nicotiana Benthamiana leaves with agrobacteria stain AGL1 and GV3101 with OD600=0.3 -0.7 could cause cell death. After injection, to some extent, high light may inhibit the programmed cell death, we speculate that low light may contribute to the accumulation of heterologous protein.
Key words:Nicotiana Benthamiana; Transient Transformation; Progammed Cell Death
目 录
摘 要 1
Abstract 1
引言 2
1.材料与方法 3
1.1材料 3
1.1.1烟草 3
1.1.2试剂 4
1.1.3仪器设备 4
1.2方法 4
1.2.1载体构建 4
1.2.2农杆菌的转化 4
1.2.3 本生烟的瞬时转化 5
2．结果与分析 5
2.1本生烟的种植 5
2.2质粒构建和农杆菌的转化 6
2.3不同光照强度对细胞坏死的影响 6
2.4不同的农杆菌和重悬液OD值对本生烟细胞程序性死亡的影响 7
3．讨论 8
参考文献 9
致谢 10
农杆菌介导的本生烟瞬时转化的影响因素探索引言
农杆菌介导的瞬时转化已广泛应用于各个方面的研究，比如中科院遗传所沈前华课题组在本生烟中进行瞬时转化验证了两个在植物抗病反应中起对抗性的转录因子MYB6和WRKY1与大麦对白粉病抗性基因MLA10之间的互作[1]；将大麦对白粉病抗性基因MLA10的不同区段和突变体瞬时转化本生烟叶片 ，研究得出一系列突变体能够不同程度的引发转化叶片的细胞坏死，其中全长MLA的突变体中MLA10 (D502V)引发的细胞坏死最为明显（图1），进一步的研究发现在细胞质中的MLA10引起细胞坏死而在细胞核内的MLA10引发大麦对白粉病的抗性，也即是MLA10所诱发的细胞死亡和抗病信号是分开的[2]。中科院遗传所的另一个课题组利用瞬时表达分析表明了转录因子MYC2抑制转录因子ORA59的表达，而MYC2的磷酸化偶联的蛋白水解对植物免疫至关重要，从而例证了植物是利用蛋白水解偶联的转录机制来调节对各种压力的应答[3]；2014年还有学者利用本生烟中农杆菌介导的瞬时转化来证实miR9863能特异的调控特定的MLA的表达水平[4]。