香蕉内生克雷伯氏菌KKWB-5强启动子片段的分离及鉴定

Isolation and Identification of Strong Promoter from Endophytic Klebsiella pneumoniae KKWB-5 of Banana

目的:旨在获得香蕉内生克雷伯氏菌KKWB-5的强启动子片段,以应用于香蕉内生工程菌的构建。方法:利用以kanr基因为报告基因的启动子探针载体pUCK在大肠杆菌Top10中克隆KKWB-5基因组DNA的启动子片段;将筛选到的高抗Kan的质粒导入KKWB-5,分别于LB和香蕉杆浸汁培养基(BSM)平板上检测它们的抗Kan水平;选择在BSM上抗Kan水平最高的片段15,检测该片段的基因间隔区15P的启动子活性,最后以gfp为报告基因来验证片段15P的启动子活性。结果:有7个抗Kan水平在2500μg/ml以上的Top10转化子;这7个质粒在导入KKWB-5后,它们在LB平板上的抗Kan水平有不同程度的增加,但在BSM培养基上则大为减弱;片段15P具有启动kanr基因的活性,且与原片段15的抗Kan水平相同;重组质粒pUCK-6-15Pgfp,以Top10和KKWB-5为宿主菌,在LB培养基上培养时,在荧光显微镜下均能发出绿色荧光;以KKWB-5为宿主菌,在BSM培养基上培养时,在荧光显微镜下也能发出绿色荧光。结论:片段15不仅在Top10中具有较强的启动子活性,而且在其供体菌KKWB-5中具有更强的启动子活性,其基因间隔区15P为主要的启动子区域,在BSM培养基上也具有较好的启动子活性,该启动子片段15P可以应用于KKWB-5内生工程菌的构建。

The aim is to obtain strong promoters from Endophytic Klebsiella pneumoniae KKWB-5 of Banana,which will be applied to construct an endophytic engineered bacterium of banana.Promoter probe plasmid pUCK whose report gene is kanamycin-resistance（Kanr） gene was used to screen promoter fragments from KKWB-5 DNA in E.coli Top10.There were 7 Top10 transformants whose resistant level to kanamycin were over 2500μg/ml.This 7 plasmids were transformed into KKWB-5,and KKWB-5 transformants were tested their resistant level to kanamycin on LB plate and banana stem medium（BSM） respectively.After the plasmids were transformed into KKWB-5,the resistant level to kanamycin of KKWB-5 transformants when cultured on LB showed various degree of increases,but when cultured on BSM it weakened to a great degree.Transformant K-pUCK-15 was the highest resistant clone to kanamycin on BSM,and then the intergenic spacer region 15P of inserted fragment 15 were tested its promoter activity by pUCK as Kanr gene for report gene and by pUCK-6 as green fluorescent protein gene for report gene.The results demonstrated that fragment 15P had the same resistant level to kanamycin with fragment 15,moreover,the Top10 and KKWB-5 transformants containing recombinant plasmid pUCK-6-15Pgfp could emit intense green fluorescence under UV excitation when cultured on LB,and KKWB-5 transformants could emit green fluorescence under UV excitation too when cultured on BSM.In conclusion,fragment 15 had strong promoter activity in Top 10,but had stronger promoter activity in KKWB-5.The intergenic spacer region 15P was the main promoter region of Fragment 15,and it had good promoter activity in KKWB-5 when cultured on BSM.Fragment 15P can be applied to construct an endophytic engineered bacterium of banana.