含质粒复制起始区ori44的苏云金芽胞杆菌解离载体的构建  

A Novel Resolution Vector with Bacillus thuringiensis Plasmid Replicon ori44

作　　者：吴岚[1] 孙明[1] 朱晨光[1] 张蕾[1] 喻子牛[1] 

机构地区：[1]华中农业大学生命科学技术学院农业微生物农业部重点实验室,武汉430070

出　　处：《生物工程学报》2002年第3期335-338,共4页Chinese Journal of Biotechnology

基　　金：国家 8 6 3计划课题 (No 2 0 0 1AA2 14 0 11和 2 0 0 1AA2 12 30 1);武汉市晨光计划 (No 96 5 0 0 0 10 37 2 2 )资助~~

摘　　要：将苏云金芽胞杆菌转座子Tn44 30的解离酶识别位点res分别插入克隆载体pRSETB和pUC19得到质粒pBMB12 0 1和pBMB12 0 2。这两个质粒分别经BamHI HindⅢ和EcoRI HindⅢ双酶切回收含res位点的小DNA片段 ,与穿梭载体pHT310 1经EcoRI HindⅢ双酶切后回收的含大肠杆菌复制起始区、氨苄青霉素抗性基因和红霉素抗性基因的 3 3kb片段连接 ,获得重组质粒pBMB12 0 3。封闭pBMB12 0 3两res位点外的BamHI和EcoRI位点后 ,得到解离载体pBMB12 0 4。将来源于苏云金芽胞杆菌库斯塔克亚种YBT 15 2 0的质粒复制起始区ori44片段插入pBMB12 0 4的两res位点之间 ,得到解离穿梭载体pBMB12 0 5。该解离载体插入壮观霉素抗性基因后电转化无晶体突变株 ,在辅助质粒所提供的解离酶作用下可发生解离消除抗性基因 ,解离频率为 10 0 % ,解离后的质粒稳定性为 93%。利用解离穿梭载体pBMB12 0The resolution recognization sites of transposon Tn4430 of Bacillus thuringiensis was inserted into cloning vector pRSET B and pUC19, resulting recombinant plasmids pBMB1201 and pBMB1202. Both of the mini res fragments, BamHI/HindⅢ fragment in pBMB1201 and EcoRI/HindⅢ fragment in pBMB1202, were ligated to the 3.3kb EcoRI/HindⅢ fragment of shuttle vector pHT3101, which contained the ori.Ec,amp r and em r antibiotic resistant genes, resulting recombinant plasmid pBMB1203. After deleted the BamHI and EcoRI sites which located ouside the two res sites, resolution vector pBMB1204 was resulted. There are multiple cloning sites between two copies of resolution sites which have the same direction. The plasmid replication origin ori44, which come from B.thuringiensis sub sp. kurstaki strain YBT-1520, was inserted into the multiple cloning sites of pBMB1204 and then resolution shuttle vector pBMB1205 was obtained. With spectinomycin resistant gene as target, it was found that the resolution rate is 100% and the stability of the resolved plasmid is 93%. Using this shuttle vector, antibiotic resistance markers and other non-B.thuringiensis DNA can be selectively eliminated after the selection of transformants by antibiotic resistance marker. This vector is very useful to solve the gene safety problem while has no effect on target gene expression.

关 键 词：质粒复制起始区 ori44 苏云金芽胞杆菌 解离载体 构建 

分 类 号：Q782[生物学—分子生物学] TQ453.5[化学工程—农药化工]