解毒酶基因的克隆及其在大肠杆菌和蓝藻中的表达(英文)

近几年来 ,在明确了杀虫药剂抗性机理的基础上 ,从杀虫药剂抗性的昆虫中分离出高抗性基因 (即解毒酶基因 ) ,将该基因克隆到表达载体 pRL 4 39上 ,得到表达载体 pRL B1,将其转化大肠杆菌HB10 1,获得了可以表达解毒酶基因的转基因工程菌株。同时构建了穿梭表达载体 pDC B1,并转化大肠杆菌HB10 1后 ,在抗生素氨卞霉素 (30 μg/mL)和卡那霉素 (30 μg/mL)平板上挑选阳性克隆 ,将阳性克隆的细胞、蓝藻和结合质粒以三亲结合转移的方式转入蓝藻。斑点杂交、Southern分析结果表明已经获得了Synechococcussp .PCC 794 2转基因工程藻。

EXPRESSION OF THE DETOXIFYING GENE B1 IN ESCHERICHIA COLI AND SYNECHOCOCCUS

We inserted the mosquito esterase B1 gene into the expression vector pRL|439,which possesses the strong promoter PpsbA.The recombinant plasmid pRL|B1 was used to transform E.coli HB101,and the positive clones were screened on LB medium plate containing 100?mg/mL ampicillin.The results of dot blotting and Southern hybridization demonstrated that these positive clones were transformed bacteria.Western blotting indicated that esterase B1 gene had been successfully expressed under the control of the PpsbA promoter in E.coli .;A shuttle verruction|B1 (pDC|B1) was constructed by inserting B1|cDNA from pRL|B1 into polycloning site of plasmid pDC|8.PDC|B1 was transferred into Synechoccus sp.PCC7942 through triparental conjugal transfer.Transformed single Synechococcus sp.PCC 7942 clone was obtained by neomycin screening,and large|scale culture in liquid medium was carried out.Results of Southern blotting proved that pDC|B1 was transferred into Synechococcus sp.PCC 7942.