摘要
用ＰＣＲ方法将广宿主范围质粒ＲＫ２中控制稳定性的片段ｐａｒＤＥ克隆到ｐＧＥＭ┐Ｔ载体上，然后插入到分泌表达载体ｐＥｘＳｅｃ１的ＢａｍＨＩ切点上，得到两个新载体ｐＺＬ２和ｐＺＬ３，其中ｐａｒＤＥ的转录方向分别与Ｔ７启动子相同和相反。经酶切物理图谱分析和ＰＣＲ扩增验证后，将ｐＺＬ２和ｐＺＬ３分别电击转化到成团泛菌３０８Ｒ中，在无抗生素的ＬＢ培养基中生长１００代后仍１００％地保留在细胞内。
Pantoea agglomerans has been widely used for biological control of many leaf diseases due to its ability of competitively colonize plant surface and the ability of producing versatile antimicrobials Recombinant DNA technology can be exploited to genetically engineer the bacterium for better efficacy of disease control Yet, many presently available plasmid vectors such as pCPP9 or pExSec1 are unstable in Pagglomerans, more than 99% of cells lost the plasmids after 100 generation of growth in LB medium The parDE fragment of RK2 was amplified by PCR with pTR102 as template and cloned into pGEMT to get pZL1 The 08kb fragment was cut and religated with BamHIcut pExSec1, a secretionexpression vector based on pUC21 Two new plasmids pZL2 and pZL3 with parDE inserted in different transcriptional orientation relative to T7 promoter were obtained and verified by physical mapping and PCR amplification pZL2 and pZL3 were transformed into Pagglomerans 308R by electroporation and found to be 100% maintained
出处
《中国生物防治》
CSCD
1999年第2期49-53,共5页
Chinese Journal of Biological Control
基金
国家"863"高技术计划
国家自然科学基金
山西省自然科学基金
关键词
成团泛菌
质粒稳定性
RK2
parDE
Pantoea agglomerans
plasmid stability
RK2
parDEin the cell after 100 generation growth in antibioticfree LB medium These plasmids were also stably maintained in cells growing in planta
