【作者】 姜鹏；

【导师】 秦松；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2003， 博士

【摘要】 海带遗传转化模型是以基因枪转化海带雌配子体，转化后诱导生成孤雌生殖孢子体。本文以lacZ报告基因染色结果显示，原有孤雌生殖途径只能实现外源基因的低效率与嵌合式表达。发展了雌（或雄）配子体转化后受精发育生成二倍体孢子体的新途径，均可实现lacZ报告基因的稳定表达。其中，雌配子体途径（转化后与正常雄配子体受精）的结果与孤雌生殖途径相仿，而雄配子体途径（转化后与正常雌配子体完成受精）可大幅提高转化效率与表达效率，有3.5%的植株实现均一性表达，提示外源基因的整合发生在单细胞阶段。沿用原有孤雌生殖途径，转化人乙肝病毒表面抗原（HBsAg）基因，在孤雌生殖孢子体中检测到HBsAg的整合与表达。定量ELISA显示表达水平为0.125-2.497 μg/（mg可溶性蛋白），平均为0.587 μg/（mg可溶性蛋白），即2.25克鲜海带的抗原表达量等同于静脉注射免疫所需15 μg抗原。继而利用雄配子体途径也实现了HBsAg在海带二倍体孢子体中的稳定表达，提示利用海带表达口服乙肝疫苗具有一定可行性。本文结果为大型海藻遗传转化提供了一条可能的新途径，即转化孢子后利用自然的发育过程生成转基因植株；实现了大型海藻中第一个目的基因的导入与表达。更多还原

【Abstract】 The original transformation model for the seaweed Laminaria japonica (Phaeophyceae) employed female gametophytes as micro-particle bombardment targets and parthenogenesis as a route to produce transgenic parthenosporophytes. By using the original model, in situ detection for lacZ expression was carried out in this thesis and resulted in low transformation efficiency and chimeric expression pattern. New approaches were developed in this study by transforming female and/or male gametophytes followed by inbreeding process. LacZ expression was detected in zygotic sporophytes generated by both female- or male-targeted transformation. The former showed similar results to parthenogenesis pathway, while the latter led to higher transformation and expression efficiency and 3.5% zygotic sporophytes were stained all-blue, implying the integration of lacZ at one-cell stage.A vaccine gene encoding hepatitis B surface antigen (HBsAg) was bombarded into female gametophytes and transgenic parthenosporophytes were obtained. Results of quantitative ELISA showed that HBsAg in transgenic kelp was 0.529 μg/mg soluble proteins on average and the highest value was 2.497 μg/mg, implying that recombinant HBsAg in 2.25 g fresh weight of transgenic kelp corresponded to 15 μg vaccine required in vein injection for one adult. Furthermore, HBsAg gene expression was obtained in zygotic sporophytes by using male gametophytes-targeted bombardment.This research provided a potential method for seaweed transformation using spore-mediated bombardment followed by normal development process, also reported for the first time the introduction and expression of a target gene in transgenic seaweed.更多还原