利用高效转座质粒构建羽毛分解菌Streptinomonas maltophilia YHJ-1的突变体库  

Construction of Insertion Mutant Library of Stenotrophomonas maltophilia YHJ-1 Degrading Feather Using Highly Efficient Transposon Plasmid

作　　者：黄亦钧[1] 季金殿[1,2] 尤升波[1] 游银伟[1] 单成纲[3] 岳寿松[1] 

机构地区：[1]山东省农业科学院高新技术研究中心,济南250100 [2]山东农业大学动物科技学院预防兽医系,泰安271018 [3]山东省农业科学院原子能研究所,济南250100

出　　处：《农业生物技术学报》2009年第5期931-936,共6页Journal of Agricultural Biotechnology

基　　金：山东省农业科学院高技术自主创新基金(No.2006YCX029);山东省农业科学院博士基金资助

摘　　要：嗜麦芽窄食假单胞菌(Streptinomonas maltophilia)YHJ-1是一株能高效降解羽毛角蛋白的菌株;该菌对卡那霉素等多种抗生素不敏感。利用携带Tn5转座酶和卡那霉素抗性转座质粒pRL27和含氯霉素抗性基因盒的pMCm质粒,构建了以氯霉素抗性作为选择标记的转座质粒pRH30。以大肠杆菌(Escherichia coli)UQ3021/pRH30为供体,通过双亲本杂交,构建了S.maltophilia YHJ-1的插入突变体文库,该库共含1246个转化子。随机挑选4个突变子经氯霉素抗性和PCR分析检测,结果显示突变体均有氯霉素抗性和氯霉素抗性基因,表明文库是可靠的。已从文库中筛选出3株不能降解羽毛转化子,表明利用氯霉素抗性的转座质粒pRH30构建菌株YHJ-1的突变体是克隆其相关基因并进行遗传分析的有效方法。Stenotrophomonas maltophilia YHJ-1, isolated from the poultry factory, shows the high feather keratin degradation activity as well as many antibiotic resistances including kanamycin.In this study, a transposon plasmid pRH30 was developed using chloramphenicol gene to replace kanamycin gene with transposon plasmid pRL27 containing both hyperactive Tn5 transposase and kanamycin resistance gene and pMCm, carrying chloramphenicol cassette.And an insertion library containing 1 246 mutants was produced with pRH30 by biparental conjugation between donor Escherichia coli UQ3201/pRH30 and recipient strain YHJ-1.The results of insertion mutants, subjected to detection of chloramphenicol resistance and PCR sequencing analysis, indicated that the library was reliable.The fact that 3 mutants were unable to degrade feather suggested that transposon mutagenesis with pRH30 was a highly efficient method for facilitating the isolation of genes and analyzing the molecular mechanism on feather-degrading of S.maltophili YHJ-1.

关 键 词：嗜麦芽窄食假单胞菌YHJ-1 转座子突变 插入突变体文库 

分 类 号：S188[农业科学—农业基础科学]