摘要
用抗性库蚊酯酶基因B1的cDNA片段插入质粒pRL439中的强启动子之后,再与穿梭表达载体pDC8相连构建成大肠杆菌蓝藻穿梭表达载体pDCB1,然后通过三亲接合转移法将pDCB1转入蓝藻Synechococcussp.PCC7942中,经新霉素筛选获遗传稳定的转基因藻株;纯化单藻落在液体中扩大培养,提取蓝藻质粒,Southern杂交确证B1cDNA已转入受体细胞;用酯酶的特异性底物β乙酸萘酯(βNA)检测B1的表达,转基因藻对βNA的降解明显高于野生藻,证明酯酶B1基因在转基因藻中得到表达。
Plasmid pRL B1 was constructed from detoxifying gene(called B1) of pesticide resistant Culex and from plasmid pRL 439 containing the strong promoter PpsbA. E.coli cyanobacteria shuttle expression plasmid pDC B1 was constructed from shuttle vector pDC 8 and from recombinant plasmid pRL B1,then it was transferred into Synechococcus sp. PCC7942 by triparental conjugative transfer.The existence of B1 was detected by Southern analysis,and the expression of B1 was confirmed by enzyme activity analysis of detoxification of transgenic cyanobacteria.Experimental results indicated that the transgenic cyanobacteria could degrade β naphthyl acetate(β NA),a specific substrate of esterase.The enzyme activity of transgenic strain was higher than that of the wild type. It may be the first report on transformation of detoxify gene of pesticide resistant culex into Synechococcus strain.
出处
《生物工程学报》
CAS
CSCD
北大核心
2000年第1期42-45,共4页
Chinese Journal of Biotechnology
基金
国家自然科学基金资助项目!( 3 96 80 0 0 1)
山东省自然科学基金资助课题!(Y98D16 0 6 4 )
关键词
解毒酶基因
蓝藻
基因表达
农药污染治理
克隆
Detoxifying gene, Synechococcus, expression,transgenic cyanobacteria,conjugative transfer
