利用转座子标签法构建埃氏巨型球菌乙酸生成关键酶基因缺失工程菌  

Constructing Megasphaera Elsdenii Mutants of Deleting Acetic Acid-Producing Key Enzyme Gene by Transposon Tagging Method

作　　者：杨文艳[1] 刘国文[1] 崔晓霞[1] 逄晓阳[1] 冯海华[1] 王哲[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《中国兽医学报》2006年第6期669-672,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金重点资助项目(30230260;30600441)

摘　　要：应用转座子标签法,通过转座子供体菌E.coliS17-1/pZJ25∷Tn5对受体菌埃氏巨型球菌进行转座子诱变,采用含卡那霉素和氟乙酸钠的选择性培养基筛选接合子,共筛选出稳定的对卡那霉素和氟乙酸具有抗性的转座工程菌9株。对埃氏巨型球菌的突变株进行16SrRNA和Tn5的PCR鉴定,及乙酸激酶(AK)和磷酸转乙酰酶(PTA)酶比活力分析,确定突变株属于pta基因缺失型氟乙酸抗性菌株。By transposon tagging method ,Escherichia coli S17-1/pZJ25：： Tn5 as transposon donator,megasphaera elsdenii as receptors of genetic engineering recombination,the Tn5 was inserted and integrated into the genome of wild type magaspkaeru elsdenii. Nine stable mutants were obtained by selective medium containing the kanamycin and baran. The 16S ribosomal RNA gene and Tn5 transposon gene of the strains TnH6 were amplified by polymerase chain reaction（PCR） and sequenced. Furthermore ,the specific activity of phosphotransacetylase （PTA） and acetate kinase （AK）, which were acetic acid-producing key enzymes in the megasphaera elsdenii,were measured. There by, the mutants were proved to be the pta gene deletion engineering bacteria.

关 键 词：埃氏巨型球菌 转座子 氟乙酸 磷酸转乙酰酶 乙酸激酶 

分 类 号：S856[农业科学—临床兽医学]