GL-7-ACA酰化酶基因片段的酶谱分析及基因定位  

Restriction Mapping of GL-7-ACA Acylase Gene Fragment and Its Localization

作　　者：杨蕴刘[1] 恽定芳 彭惠琳 焦瑞身[1] 

机构地区：[1]中国科学院上海植物生理研究所

出　　处：《生物工程学报》1992年第1期15-22,共8页Chinese Journal of Biotechnology

摘　　要：本文报道了从假单胞菌130菌株(Pseudomonas sp.130)染色体上克隆得到的6.8kb的GL-7-ACA酰化酶基因片段的限制酶谱,基因定位以及在不同的大肠杆菌基因启动子控制下酰化酶基因的表达水平。结果表明,所克隆的片段上,不存在EcoR Ⅰ、Hind Ⅱ、Cla Ⅰ切点,分别具有一个Hpa Ⅰ、两个Xho Ⅰ、三个BamH Ⅰ以及四个Pst Ⅰ切点,同时初步确定了这些酶切位点之间的相对位置。经过一系列次级克隆研究,GL-7-ACA酰化酶基因已被定位在3.0kb的B_2-B_3-Hpa Ⅰ片段上。实验比较了以pACYC184、pDR540、pUC10等为载体的次级克隆株(分别为pMR9、pMR10和pMR11)在大肠杆菌中酰化酶基因的表达水平,测定数据表明tac启动子的启动活力比tet启动子强,即pMR10的产酶量比pMR9高一倍,而当tac启动子前再串接一个lac启动子时(pMR11),产酶水平并不进一步提高。本文还对假单胞菌基因在大肠杆菌中的表达进行了讨论。This paper presents the results about the restriction mapping of recombinant plasmids pMR5 and pMR6 containing GL-7-ACA acylase gene from Pseudomonas sp. 130, gene localization and its expression under the control of different promoters, tet, tac or Iac/tac, in Escherichia coli. The analysis of gel electrophoresis of pMR 5 cleaved with several kinds of restriction enzymes indicated that there is no sites of EcoR Ⅰ, HindⅢ and Cla Ⅰ but the presence of following sites: one Hpa Ⅰ, two Xho Ⅰ, three BamH Ⅰ and four Pst Ⅰ on the cloned gene fragment. The restriction maps of pMR 5 and pMR 6 were determined by comparative digestion of various endonucleases. The gene of GL-7-ACA acylase was localized on a 3.0kb fragment of B_2-B_3-Hpa Ⅰ from the studies on a serial subcloning. Expression of subclones pMR9, pMR10 and pMR11 in E. coli was compared. Higher yield of acylase was obtained when the gene fragment was placed downstream of the tac promoter. The expression of Pseudomonas gene in E. coli was also discussed.

关 键 词：基因定位 基因表达 GL7ACA酰化酶 

分 类 号：Q78[生物学—分子生物学]