甲基营养菌MP688中启动子探针载体的构建  

Development of a Promoter Probe Vector for Methylovorus sp. MP688

作　　者：杜宝华[1,2] 葛欣[1] 王文溪[1,2] 熊向华[1] 汪建华[1] 何建勇[2] 张惟材[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]沈阳药科大学,辽宁沈阳110016

出　　处：《生物技术通讯》2012年第5期644-647,共4页Letters in Biotechnology

基　　金：国家科技支撑计划(2007BAI46B01);国家高技术研究发展计划(2006AA020303)

摘　　要：目的:以半乳糖苷酶基因作为报告基因,构建适于探测甲基营养菌MP688启动子的载体。方法:通过PCR扩增半乳糖苷酶基因片段,连入质粒pCM66构建启动子活性探针载体pMPlacZ。根据MP688的基因组序列设计引物,通过PCR扩增5个pqqA基因、核糖体亚基A基因(rpsA)、核糖体亚基B基因(rpsB)、伴侣分子groel基因和甲醇脱氢酶基因(mdh)等共9个基因的启动子序列,将这9个启动子片段连入pMPlacZ进行活性测定。结果:构建了一个适于探测甲基营养菌MP688的启动子活性的载体pMPlacZ;利用构建的报告系统对MP688中9个基因的启动子进行了活性比较,得到3个与吡咯喹啉醌合成相关的强启动子。结论:构建的启动子活性检测载体可以有效、灵敏地用于MP688强启动子的筛选和启动子活性检测。Objective： To develop a versatile probe vector used for promoter evaluation in Methylovorus sp.MP688.Methods： The fragment of β-galactosidase coding sequence was ligated into pCM66,generating probe vec tor pMPlacZ.Nine promoter sequences,including rpsA,rpsB,groel and mdh genes as well as five pqqA genes,were amplified from MP688 genome DNA.The nine fragments were inserted into pMPlacZ and β-galactosidase ac tivity was determined in different conditions.Results： pMPlacZ was demonstrated to be a versatile and sensitive probe vector for promoter evaluation in Methylovorus sp.MP688.By using the probe vector,three strong promoters which were closely relative to pyrroloquinoline quinone（PQQ） synthesis were isolated and identified.Conclusion： pMPlacZ can be used for promoter screening and enzyme activity determination.

关 键 词：甲基营养菌 半乳糖苷酶 启动子活性检测载体 启动子活性测定 

分 类 号：Q78[生物学—分子生物学]