产杀菌蛋白菌株A35的基因库构建及细菌强启动子活性片段的克隆  

Construction of Genomic Bank of Bacteriocinogenic Strain A35 of Pseudomonas Solanacearum and Molecular cloning of Bacterial Strong Promoter DNA Segments

作　　者：赵立平[1] 梁宏[1] 黄静[1] 杜建中[1] 杨宝茹 王卉[1] 刘少翔[1] 张建华[2] 郑继法[2] 

机构地区：[1]山西省农业科学院作物遗传研究所 [2]山东农业大学烟草研究室

出　　处：《山西农业科学》1993年第1期2-7,共6页Journal of Shanxi Agricultural Sciences

基　　金：山西省自然科学基金资助

摘　　要：青枯假单胞菌无致病力突变株 A35可产生对蛋白酶 K 处理及热处理敏感的蛋白类杀菌物质——细菌素,制成活菌制剂用于种子、根系处理,有一定的防病效果。作为分离杀菌基因的基础,用载体 pLAFR3构建了 A35的基因文库,得到1138个克隆,插入片段的平均长度22.63kb,有99%的概率保证每个基因至少在库内出现1次。从库中得到1个在28℃下大量产生胞外多糖的克隆。作为提高拮抗基因表达强度的基础,用启动子探针质粒 pIJ3100克隆软腐欧氏杆菌的基因调控区,得到6400个克隆的启动子片段文库,通过三亲交配法转移到软腐菌中得到2300个接合子,通过筛选,得到4个抗560μg/ml 氯霉素的克隆。酶切分析表明,重组质粒均有外源 DNA 片段的插入,其中pSLB1的插入片段长度约1.65kb,左端为 BamHⅠ和 EcoRⅠ切点,右端是 HindⅢ切点。对该片段的亚克隆正在进行,以便用于构建高强度表达的杀菌基因。Molecular work was conducted in order to optimise the effects of biological control with the bacterioeinogenic strain A35 of Pseudomonas solanacearum.High-molecular weight genomic DNA of A35 was purified and partially digested with EcoRI,15-35kb long fragments pooled and ligated with EeoRI-cut,phosphatase-treated PLAFR3 DNA.The ligation reaction was pakaged in vitro and was used to transfect E.coli DH5a.1138 white transformants were obtained on LB+IPTG+Xgal plates. Plamid screening showed that 19/20 randomly-selected clones were bonafide recombinants which have an average insert size of 22. 63kb.It was concluded that each P.solannacearum gene should ap- pear in the bank at least once With the probability of over 99%.As a possible route to increase the ex- pression of bacteriocin gene,molecular work was conducted to isolate DNA fragments with strong pro- moter activities.Chromosomal DNA of Erwinia carotovora strain ER10 was digested by Sau3A and ligated with BamHI digested promoter probe plasmid pIJ3100.The ligation was used to transform E. coli ED8767.Transformants were obtained and conjugated with ER10.Four highly Crn resistant clones were isolated,which could survive in medium containing Cm antibiotics as high as 560mg/l, while over 99% transformants were resistant to Cm at a concentration of 25mg/l.Physical mapping showed that,all 4 clones had foreingn DNA inserts,among these,PSLB1 had an insert of 1.65kb, having unique BamHI site on the left end and HindⅢ site on the right.Further characterization of the plasmid is being undertaken

关 键 词：青枯假单胞菌 细菌素 基因文库 

分 类 号：S482.291[农业科学—农药学]