【作者】 王昌正；

【导师】 晏月明； 胡赞民；

【作者基本信息】 首都师范大学 ， 分子遗传学， 2003， 硕士

【摘要】 本实验采用农杆菌介导法和基因枪介导法，研究了乙肝表面抗原基因(HBsAg)导入可生食的莴苣(生菜) (Lactuca sativa)的核基因组和叶绿体基因组，并期望获得转基因莴苣植株，从而建立以莴苣为生物反应器生产可食用的乙肝疫苗。研究结果如下： (1)将乙肝表面抗原基因(adw型)与细胞核载体PB、PBG以及叶绿体表达载体PLCTB进行定向克隆，经PCR和测序鉴定都获得了重组子，分别命名为PB-adw、PBG-adw、PLCTB-adw。其中PB-adw含烟草花叶病毒CaMV35S组成型启动子，PBG-adw含马铃薯蛋白酶抑制剂Ⅱ基因的损伤诱导型启动子PIⅡ，PLCTB-adw含有莴苣启动子与终止子，并在目的基因两侧设有叶绿体同源片段。 (2)转基因莴苣筛选压力浓度的确定，卡那霉素(Kanamycin)为100mg/L，壮观霉素(Sepctinomycin)为20mg/L时能有效的将转化和非转化植株筛选出来。 (3)细胞核载体PB-adw、PBG-adw均采用农杆菌介导法将adw导入莴苣，细胞核转化获得了生长良好的抗性莴苣植株，经PCR、PCR-Southern、southern斑点杂交分析证实，adw基因已整合到莴苣基因组中。其中，PB-adw转化率高于PBG-adw，说明莴苣转化中CaMV35S启动子优于PIⅡ启动子。 (4)叶绿体表达载体PLCTB-adw采用了基因枪介导法将adw导入莴苣。由于叶绿体转化植株生长较为缓慢，筛选周期比细胞核转化长，并且一段时间后会出现白化现象。故实验停留在抗性小苗获得的结果，还没有进行下一步的检测，具体原因有待进一步分析。更多还原

【Abstract】 In this experiment, HBsAg (adw subtype) was introduced into lettuce(Lactuca sativa) with the Agrobacterium transformation system and biolistic method. We hope to obtain the transgenic lettue with HBsAg so that it can be used as edible vaccine. The experiment results covered several following points:(1) adw was cloned into nuclear plasmid PB, PBG and chloroplast expression plasmid PLCTB . The expression vectors with adw, namely PB-adw, PBG-adw, PLCTB-adw were constructed by gene recombination technology.(2) For screening the transgenic lettuces from the whole materials, suitable concentration of kanamycin was 100mg/L and spectinomycin was 20mg/L.(3) Regenerated plants with kanamynic resistantance were obtained. PCR , PCR-Southern blot analysis , southern dot blot analysis of lettuce DNA confirmed that adw gene had been integrated into the plant genome. The results also showed that the transformation frequency of PB-adw was higher than that of PBG-adw, which suggested that CaMV35S promoter would be better than PI II promoter in the case of transgenic lettuce.(4) The chloroplast expression plasmid PLCTB-adw, which contained lettuce psb promoter and terminor, besides two homogeneous sequences at adw gene flanks was used to transforme lettuces by the biolislic method. Because selective period of chloroplast transformation was longer than that of the nuclear transformation, only the several weakly regenerated plants with spectinomynic resistantance were obtained. Further screening and molecular detections need to be done.更多还原