摘要：大豆花叶病毒病是危害大豆产量和品质最为严重的病害之一。虽然几年前大豆抗大豆花叶病毒的主要抗病位点都已经通过遗传作图定位在相应的染色体区段内，但由于大豆转化目前普遍存在转化效率低、转化植株嵌合体比例高、转化周期长等问题，使得最终确定抗病基因的工作进展缓慢。抗病基因介导的植物免疫反应通常引起过敏性反应，进而导致细胞死亡。本实验试图依据这一现象，建立通过原生质体瞬时表达技术实现抗病候选基因筛选的方法。在本实验中，我们构建了基于gateway技术的瞬时表达载体并以此为基础构建了报告基因GUS及效应因子Avh241的过表达载体，提取了大豆叶片原生质体，但PEG介导的大豆原生质体的质粒转化暂未成功；最后本文总结了实验失败的可能原因，为下一次的实验尝试奠定基础。29878
毕业论文关键词：原生质体；瞬时表达；抗病基因；过敏性反应
Establishment of Resistance-related Gene Identification Method by Gene Transient Expression within Soybean Protoplast
Abstract：Soybean mosaic virus disease is one of the most serious diseases that endanger soybean yield and quality. It is difficult to identify the disease resistance gene due to the defects that low transformation efficiency and time-consuming of soybean genetic transformation though the major soybean mosaic virus resistance locus have been fine mapping on corresponding chromosomal. Disease resistance gene-mediated plant immune response usually elicits hypersensitive response, even leading to cell death. This phenomenon may serve as the basis for identifying disease resistance response. Thus we attempt establish the method for resistance-related gene identification through soybean protoplast base on this phenomenon. On this purpose we constructed a transient expression vector based on gateway technology, and constructed the overexpression vector of reporter gene GUS and pathogen effectors Avh241. We also isolated soybean protoplast successively. Unfortunately, we failed to transform plasmid into the protoplast efficiently. Finally, we summarized the possible causes for the failure of the experiment, which will increase the chance for success at the next attempt.
Key words: Protoplast; transient expression; Disease resistance gene; hypersensitive response
目 录
摘要1
关键词1
Abstract1
Key words1
引言2
基于gateway系统的瞬时表达载体的构建 4
1材料与方法 4
1.1材料 4
1.2方法 4
1.2.1引物的设计 4
1.2.2 gateway表达盒的扩增及检测 4
1.2.3 gateway表达盒PCR产物的纯化5
1.2.4 gateway表达盒与pMD20载体的连接5
1.2.5 连接产物的转化与涂板 5
1.2.6 单克隆的检测 6
1.3 实验结果 6
1.3.1 引物的设计6
1.3.2 gateway表达盒的扩增及检6
1.3.3 单克隆的检测 7
1.4 讨论8
Avh241及GUS表达载体的构建8
2.材料和方法8
2.1实验材料 8
2.2.1 pMDEG的摇菌及质粒提取步骤 9
2.2.2 pDonr-Avh241及pDonr-GUS与pMDEG载体的LR反应 9
2.2.3 连接产物的转化与涂板 9
2.2.4 单克隆的检测 9
2.3 实验结果 9
2.3.1 pMDEG的摇菌及质粒提取 10
2.3.2 单克隆的检测10
2.4 讨论10
大豆原生质体的提取及转化 11
3材料和方法11
3.1 实验材料11
3.2 实验方法11
3.2.1大豆原生质体提取及转化配方 11 利用大豆原生质体瞬时表达技术鉴定大豆抗病相关基因方法的建立:http://www.youerw.com/shengwu/lunwen_25317.html