依纽小单孢菌接合转移体系的构建  

Construction of Conjugal Transfer System of Micromonospora inyoensis

作　　者：张国华[1] 洪文荣[1] 沈剑锋 樊伟明 

机构地区：[1]福州大学生物科学与工程学院,福州350108 [2]浙江震元制药有限公司,绍兴312000

出　　处：《生物技术通报》2011年第11期216-220,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31070093/30801449);福建省教育厅科研项目(2007F5070)

摘　　要：以依纽小单孢菌HP变种基因组DNA为模板,扩增位于西索米星3',4'-双脱羟基酶基因sisI上下游序列的两端同源交换臂,在两臂之间添加抗性筛选标记ermE基因,并在该基因上游加入组成型强启动子ermE*,以强化筛选标记。将该外源DNA序列插入到质粒pKC1139,构建重组质粒pFD57。转化大肠杆菌ET12567后,经接合转移导入依纽小单孢菌中,经抗性筛选得到两株阳性菌株,命名为HP-I-1和HP-I-2。经PCR验证和测序,结果表明,重组质粒已整合到染色体DNA上。依纽小单孢菌接合转移体系的构建达到了预期目的,并实现了对该体系的优化。Two exchange arms,which were respectively located upstream and downstream of gene sisI encoding 3′,4′-double dehydroxylase,were amplified by PCR from Micromonospora inyoensis HP mutant genomic DNA.The marker gene ermE and constitutive promoter ermE＊ was then inserted between the arms.With these fragments,we constructed pKC1139-derived vector pFD57.The recombinant vector was transformed to Escherichia coli ET12567（pUZ8002）,and then transfered to Micromonospora inyoensis HP mutant by conjugation.Two positive transformants were selected from the plate with erythromycin,which were named as HP-I-1 and HP-I-2 respectively.After PCR identification and sequencing,it confirmed that pFD57 was integrated into the chromosome of Micromonospora inyoensis HP mutant.

关 键 词：DNA重组 分子遗传学 依纽小单孢菌 sisI基因 

分 类 号：Q78[生物学—分子生物学]