洋葱伯克霍尔德菌T1828和ZWL15-Tn5转座子插入突变体库的构建及相关突变体的筛选初探

doi:10.11924/j.issn.1000-6850.2013-1710

中国农学通报 ›› 2014, Vol. 30 ›› Issue (9): 242-247.doi: 10.11924/j.issn.1000-6850.2013-1710

所属专题：园艺

洋葱伯克霍尔德菌T1828和ZWL15-Tn5转座子插入突变体库的构建及相关突变体的筛选初探

刘好桔钟义军饶志强葛岚吴晓玉

收稿日期:2013-06-21 修回日期:2013-10-29 出版日期:2014-03-25 发布日期:2014-03-25
基金资助:
江西省自然科学基金“质膜流动性对冠毒素二元信号系统感应蛋白的影响”(2008GJN0016）；江西农业大学校基金项目“伯克霍尔德菌株BCC930的冠毒素生物合成基因的初步定位与分子鉴定”(2548）。

Construction of Mutation Library in Burkholderia cepacia by Inserting Tn5 Transposon and the Screening of Relevant Strain

Received:2013-06-21 Revised:2013-10-29 Online:2014-03-25 Published:2014-03-25

摘要/Abstract

摘要： 采用双亲结合法将含有Tn5转座子的质粒pRLl063a导入洋葱伯克霍尔德菌T1828和ZWL15中，在含有卡那霉素和链霉素的抗性平板上筛选抗性接合子。通过随机插入诱变后，共获得300个突变株。PCR检测随机挑选的6株突变株，结果显示为阳性；且6株突变株发酵后高效液相色谱检测不能合成冠毒素。研究结果表明，6株突变株均已被Tn5插入染色体基因组且丧失冠毒素合成的能力。本研究为今后转座子插入位点侧翼序列的克隆与测序，并进行功能回复试验提供了基础。

关键词: 体外培养, 体外培养

Abstract: The plasmid pRLl063a containing Tn5 transposon was transferred into Burkholderia cepacia strains T1828 and ZWL15 by conjunction, and associators were screened on restrictive plates with kanamycin and strepmycin. 300 mutants were obtained by Tn5 randomly inserted. PCR test were positive for six randomly selected mutants, and the mutants could not synthesize coronatine by HPLC detection. The results showed that, the genome for six randomly selected mutants had been inserted by Tn5 and lose the ability to synthesize coronatine. This study provided a basis for cloning and sequencing the transposon insertion sites’flanking sequence and the recovery test function.

刘好桔钟义军饶志强葛岚吴晓玉. 洋葱伯克霍尔德菌T1828和ZWL15-Tn5转座子插入突变体库的构建及相关突变体的筛选初探[J]. 中国农学通报, 2014, 30(9): 242-247.

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洋葱伯克霍尔德菌T1828和ZWL15-Tn5转座子插入突变体库的构建及相关突变体的筛选初探

Construction of Mutation Library in Burkholderia cepacia by Inserting Tn5 Transposon and the Screening of Relevant Strain

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