【作者】 申慧峰；

【导师】 张大兵；

【作者基本信息】 宁夏大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 为研究口蹄疫病毒(FMDV)表面抗原VP1在植物中的表达,分离克隆了“O”型FMDV表面抗原VP1基因,以马铃薯块茎特异性表达的Patatin启动子分别构建了2个植物双元表达载体pBIVP和pBICVP。pBIVP含有Patatin启动子和马铃薯蛋白酶抑制子基因Ⅱ(Pin2)的3’末端非翻译区(UTR)序列,在VP1基因的末端融合了编码内质网滞留信号肽(SEKDEL)核苷酸序列;pBICVP中除了含有Patatin启动子和Pin2的3’末端非翻译区外,在VP1的5’端融合了霍乱毒素B亚基基因(CTB),3’末端融合了编码SEKDEL酸序列。通过农杆菌介导分别将这2种不同构建的VP1转入马铃薯,获得卡那霉素(Km)抗性的幼苗。PCR检测结果表明VP1已转入马铃薯,转化效率达到95%。更多还原

【Abstract】 Expression foot and mouth disease virus epitope VP1 in transgenic Potato (Solatium tuberosum)To highly produce VP1 epitope of foot and mouth disease virus (FMDV) in transgenic plants, a VPJ gene of "O" serotype FMDV was cloned. Then two plant binary vectors, pBIVP and pBICVP were constructed. In pBIVP, VP1 was placed under the control of the patatin promoter which only has activity in potato tubers. The 3’ end of VP1 was fused with the hexapeptide ER (endoplasmic reticulum) retention signal (SEKDEL), and then fused with 3’ end of potato proteinase inhibitor II gene (PIN2). Similar to pBIVP, VP1 was driven by patatin promoter, directed ER(endoplasmic reticulum) retention signal peptide, and terminated by 3’ UTR of Pin2 in pBICVP . In addition, the VP1 was fused with the CTB gene in pBICVP. The two constructs were introduced into potato via Agrobacterium-mediated transformation, respectively. Results of PCR analysis of kanamycin resistant plants indicated that VP1 has been transferred into potato,and transformation efficiency was about 95%.STUDY A NEW CHLOROPLAST EXPRESSION SYSTEMChloroplast expression system is a new expression system which offers a number of unique advantages including high level of transgene expression, multi-gene expression in single transformation event and transgene containment due to maternal inheritance. However, the homologous recombinant plastid DNA of transgenic plant is the time cosuming using the traditional selection method. Also we proposed the wild type need plastid DNA could not fully removed using the antibiotics selection method, thereafter, results in unstable expression of target protein in the transgenic plant.To overcome the shortcoming of the long period be used to get the stable transgenic plants, interaction between Barnase and Barstar were introduced in construction of a novel chloroplast expression system. A novel chloroplast expression vector containing Barstar gene was constructed, and used for transformation of tobacco using microprojectile bombardment. After bombardment, transgenic plants were obtained after three months.Meanwhile, a plant binary vector, which containing Barbase gene with chloroplast transporting signal peptide, was constructed and introduced into tobacco via Agrvbacterium-mediated transformation. The selection and analysis of transgenic plants were going on.更多还原