第九章 生物技术在药用植物育种上的应用教学教材.ppt

一些常见的遗传选择标记和报告基因（Reporter Genes/Selectable Markers） Neomycin Phosphotransferase gene(nptII) 抗卡那霉素 Hygromycin(hpt) 抗潮霉素 Phosphinothricin(bar) 抗除草剂 GUS β-glucuronidase enzyme which converts colorless substrate to blue product β 葡萄糖苷酸酶基因 Luciferase (firefly) GFP 绿色荧光蛋白基因 目前主要采用两种质粒基因转化载体系统，一元载体系统和二元载体系统，目前在植物转化中大多是二元载体系统。 Co-integrative 一元共整合载体 Binary vector 二元载体 LB RB 用于植物转化的工程质粒pCAMBIA1300图谱 pB19hpc (Golden Rice Cassette) TL TR aphIV 35S Gt1 psy 35S rbcS crtl Hygromycin Resistance Phytoene Synthase Phytoene Desaturase T-DNA Border T-DNA Border Selectable Marker Gene of Interest Gene of Interest Insertion Sequence Insertion Sequence 用以转化的载体部分结构。 （三）植物遗传转化方法 1、农杆菌介导的遗传转化 2、基因枪介导的遗传转化 3、花粉管通道法 1、农杆菌介导的植物遗传转化 农杆菌介导法植物转基因的原理 Agrobacterium-mediated plant transformation Agrobacterium tumefaciens mediated plant transformation 农杆菌介导的植物遗传转化必须具备2个前提： 1、植物能够被农杆菌侵染 单子叶不易侵染，双子叶植物容易侵染。 2、植物比较容易再生 regenerable cells Transformed cells Cells containing new DNA that are able to regenerate into a new plant 2、基因枪转化法(biolistics) uses a ‘gene gun’ DNA is coated onto gold (or tungsten) particles (inert) gold is propelled by helium into plant cells if DNA goes into the nucleus it can be integrated into the plant chromosomes cells can be regenerated to whole plants The Gene Gun PDS1000 Microparticle Delivery System Helium chamber Rupture disk Macrocarrier DNA coated gold particle Stopping screen Focusing device Target tissue 基因枪转化方法在单子叶植物如玉米、水稻等用的较多。 （四）转化细胞的筛选及转基因植株鉴定 1、抗性筛选 2、报告基因 3、核酸杂交 4、PCR检测 转基因水稻根、茎和叶的GUS组织化学分析 A B C a b c a b c a b c A, 根系； B, 茎杆；C, 叶片。a, Gus 基因由35S启动子驱动； b, Gus 基因由蔗糖合酶基因RSuS1的启动子序列驱动； c, 未转基因水稻植株。 A, roots; B, stems; C, leaves. a and b represent the Gus gene is drived by 35S and RSuS1 promoter respectively; c, no-transformed plants as negative control. 以GFP为报告基因的转基因植株和气孔 Southern Blot Begin by cutting DNA of putative transgenic plant into fragments with Restriction Enzymes. Run DNA on an agarose gel “Blot