抗真菌和抗除草剂基因多价植物表达载体构建及对烟草遗传转化的研究

Construction of plant expression vectors containing two anti-fungal and one anti-herbicide genes and their expression in transgenic tobacco

利用基因重组技术,将木霉几丁质酶基因(Chi)和-β1,3-葡聚糖酶基因(Glu)进行融合并将其引入含bar基因的载体pAHC25中,成功构建了由Ubiquitin启动子分别驱动Chi-linker-Glu和bar的中间载体,然后将其上的Ubi-Chi-linker-Glu-nos和Ubi-bar-nos表达盒引入pBI121中,获得兼具除草剂抗性基因bar和真菌抗性基因Chi与Glu的三价植物表达载体pBIb-CG,并对烟草进行遗传转化,经PCR及Southern检测,共获得转基因烟草32株。外源基因已整合到烟草基因组中。离体抑菌试验表明转基因烟草叶片提取液对木霉菌和镰刀菌表现出一定的抗性。

A Chitinase gene （Chi） and a β-1, 3-glucanase gene （Glu） were amplified from pET/Chi and pET/ Glu plasmids respectively, and connected by eight neutral amino acids, to form a fusion gene （Chi-linker-Glu） which was inserted into pAHC25 by replacing the GUS gene. The expression cassettes Ubi-Chi-linker-Glu-nos and Ubi-bar-nos from recombinant plasmid pAHC25-C-G were inserted into vector pBI121 to obtain plant expression vector pBIb-CG. Leaf discs of tobacco were infected by Agrobacterium tumefaciens strain EHA105 containing this plant expression vector. The fusion gene Chi-linker-Glu was shown to have been integrated into the tobacco genome by PCR and Southern blotting analysis. In vitro testing of transgenic plants showed that they had resistance to Tricho derma viride and Fusarium solanif sppisii. The fusion gene Chi-linker-Glu was therefore expressed efficiently in transgenic tobacco to give disease-resistance.