苎麻生长素结合蛋白ABP1植物干扰表达载体的构建与检测

苎麻生长素结合蛋白ABP1植物干扰表达载体的构建与检测(9000字)
摘  要：本试验以植物双链干涉载体pFGC5941为基础，根据已克隆的苎麻ABP1基因序列和pFGC5941 反义和正义区的酶切位点，分别设计引物，用已构建的含ABP1 基因的pMD18-ABP1 质粒为模板，PCR 扩增以ABP1基因起始密码子ATG 开始的长度约为570 bp 的核心序列，构建ABP1特异片段正反向重复结构的RNAi干扰载体pFGC-ABP1，转化E.coli InVαF′，经Kan抗性筛选、酶切检测及菌落PCR验证，苎麻生长素结合蛋白ABP1植物干扰表达载体构建成功，为探讨ABP1在苎麻生长及纤维发育中的作用机理奠定基础。
关键词： 苎麻；ABP1；PCR扩增；干扰表达载体

Detecting and constructing ABP1 plant Expression Vector of ramie
Abstract：This experiment is based on plant double-stranded pFGC5941 carrier interference, according to the cloned gene sequence of ABP1 gene and the pFGC5941's restriction Enzyme cutting site in Sense Strand and Antisense Strand. designs the primers,use the plasmid pMD18-ABP1 that contains gene ABP1 as template, PCR amplificate the core sequence that about 570 bp ,it's initiation codon is the ATG the same as the gene ABP1, construct the specific segment ABP1's repetitive structure of reverse RNAi interference carrier pFGC, transformate the E.coli InVαF′, through the analysis like Kan resistance , the screening test and verify cut colony PCR. ramie auxin ABP1 plants with protein expression vector, constructing interference lay the foundation to the exploration of ABP1 ramie growth and fiber in developing mechanism .
Key words：Ramie；Auxin-binding Proteins 1；PCR amplification；interference expression vector
 

目    录
摘  要    1
关键词    1
1  前  言    2
2  材料和方法    5
2.1  材料    5
2.1.1  菌株和质粒    5
2.1.2  试剂    5
2.1.3  培养基    5
2.1.4  仪器    5
2.2  试验方法    6
2.2.1  干扰表达载体的正向构建及检测    6
2.2.2  干扰表达载体的反向构建及检测    9
3  结果与分析    12
3.1  干扰表达载体的正向构建及检测    12
3.1.1  ABP1质粒PCR扩增结果及正向构建图谱    12
3.1.2  APB1 PCR扩增产物与pFGC5941载体酶切结果    13
3.1.3正向构建的ABP1重组分子菌落PCR检测    14
3.2干扰表达载体的反向构建及检测    14
3.2.1 ABP1质粒PCR扩增结果及反向构建图谱    14
3.2.2 APB1 PCR扩增产物与pFGC5941- APB1（正向）载体酶切结果    15
3.2.3 RNAi干扰载体pFGC-ABP1菌落PCR检测    16
3.2.4  pFGC-ABP1重组载体双酶切检测    16
4    讨论    16
4.1  PCR产物酶切片断与质粒直接酶切片断的载体连接效果比较    16
4.2  连接体系的确定    17
4.3    梯度PCR    17
4.4    T4连接酶    17
参考文献    17
致    谢    19