【作者】 倪志华；

【导师】 陈亮；

【作者基本信息】 厦门大学 ， 细胞生物学， 2005， 硕士

【摘要】 植物疫苗具有生产简单、成本较低、使用安全方便、易贮存等优点,是目前植物基因工程的一个研究热点。本实验目的是将O型和A型口蹄疫的抗原决定簇融合基因O21- O14 -A21-HBcAg通过农杆菌介导法转入粳稻品种日本晴中。目前已建立起高效转化体系,经组织化学及分子生物学方法检测确认获得的再生植株为转基因株系,RT-PCR检测结果表明融合基因已正常转录,并获得了子一代的转基因植株。主要实验结果如下:(1)建立日本晴的遗传转化体系:确定了农杆菌介导的外源基因转入日本晴的最佳转化条件。实验认为在愈伤组织经过干燥预处理,农杆菌液OD600约为0.6,共感染时间为20 min,共培养培养基中AS浓度为50μmol/L的条件下,能够获得较高的转化率。(2)转基因株系的获得:获得207棵再生植株,检测到193棵为转基因株系。再生植株根和叶片经组织化学染色检测到GUS基因的表达,PCR、斑点杂交及Southern杂交证实了外源基因已整合到日本晴基因组中。(3)转录水平的检测:通过RT-PCR检测,判断目的基因已经正常转录。(4)转基因株系子一代的获得:获得52个转基因株系的种子总共892粒,将部分种子萌发,培育成小苗,经组织化学染色和PCR鉴定,证实外源基因已遗传至子一代。更多还原

【Abstract】 Plant-derived vaccine is becoming one focus of plant gene engineering, which has such advantages as easy production, low cost, convenient and safe use and easy storage. In this experiment, fused gene O21- O14 -A21-HBcAg containing both type O and A FMDV antigenic determinants were introduced into rice (Oryza Sativa Japonica cultivar Npponbare ) mediated by Agrobacterium.Up till now an effective transformation system has been made, and 193 transgenic seedlings were obtained. PCR, Southern dot blotting, PCR-Southern blotting and GUS gene expression detection has demonstrated their reliability. RT-PCR show the fused gene has been transcribed in the rice. All the work was summarized below:(1) Established the genetic transformation system of Npponbare: The highest transformation frequency was obtained when the transformation process carried at callus pre-dried, Agrobaterium solution concentration reach 0.6 at OD600, the infected time longer than 20min, AS concentration reach 50μmol/L of co-culture medium.(2) Obtain of transgenic seedlings: Altergether there were 207 regeneration seedlings, via PCR, dot blotting, 193 seedlings were considered to be transgenic seedlings. Then the GUS gene expression was confirmation by histochemistry dying. All these reveal that the fused gene had inserted into the genome of Npponbare.(3) Detetion on transcript level: RT-PCR show the fused gene has been transcribed in rice .(4) Obtain of transgenic sub-generation seedlings: Altergether there were 892 seeds of 52 transgenic seedlings.Partial seeds bourgeoned and transgenic sub-generation seedlings were obtained.更多还原