利用烟草和拟南芥建立BSFE生物反应器-生物化学与分子生物学专业毕业论文.docxVIP

摘 摘 要 枯草芽孢杆菌纤溶酶是枯草芽孢杆菌N18分泌的碱性丝氨酸蛋白水解酶，临床上 可以用于预防和治疗血栓栓塞性疾病，可用于开发新型溶栓药物。 本研究克隆了枯草芽孢杆菌纤溶酶基因(Bacillus subtilis fibrinolytic enzyme， BSFE)及其前导肽序列。构建了微生物表达载体和植物表达载体。采用农杆菌介导转 化野生型烟草和野生型拟南芥，PCR鉴定转基因植株，并利用纤维蛋白平板法检测 BSFE纤溶活性。通过大肠杆菌表达了枯草芽孢杆菌纤溶酶蛋白：．采用农杆菌介导转 化获得转BSFE基因烟草和拟南芥，PCR检测证实BSFE基因已在转基因烟草和转基 因拟南芥基因组内整合，并且通过纤溶活性检测进一步证实BSFE可在转基因烟草和 转基因拟南芥体内表达，并可通过组织及根系分泌型表达，建立了植物组织及根系分 泌表达外源蛋白的系统模型。 该研究为后续微生物发酵生产和产业化以及进一步阐明外源蛋白分泌表达机理 和建立植物根系分泌生物反应器提供依据。 弓g鑫鼎枯草芽孢杆菌纤溶酶；农杆菌：遗传转化；烟草；拟南芥；分泌生物反应器 Establishment Establishment of BSFE Bioreactor in Nicotiana tabacum and Arabidopsis thaliana Abstract Bacillus subtil捃fibrinolyfic elizyme(BSFE)is all alkaline锨ine proteinase produced by Bacillus sub打lis N1 8，which Call be applied clinically弱the third generation of thrombolysis medicine to prevent or to cure diseases related to thrombus，and has a promising market prospect． In this study,the gene ofBSFE and its signal peptide sequence were cloned,and Were subeloned into the E coli expression vector and plant expression vector．BSFE gene and its signal peptide were introduced into wild-type Arabidopsis and tobacco by agro-bacterium mediated transformation．Transgenic plants were identified by PCR and BSFE fibrinolysis activity was assayed on the plates with swine fibrin． The results showed that the BSFE could be expressed by the host E．coli．And both the transgenic Arabidopsis and tobacco could secret target protein from leaf,stem Or root．A model of expression and secretion of foreign proteins by plant tissue and root system were established,which provides some refferenees for future fermentation with dustrialized production of foreign proteins． Key words：Bacillus subtilis fibrinolytic enzyme(BSFE)：Agrobacterium tumefaciens： genetic transformatio：Arabidopsis thaliana；Nicotiana tabacum；Secreting type Bioreactor Di rected by：Prof．WANG RuiGang Prof．LI Guoding App I i cant for Master degree：WANG Fu—Hu i(Master ofBiochemistry mad Molecular Biology) (College ofBioengineering,Inner MongoliaA班cultural University,