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| Isolation and Identification of Strong Promoter from Endophytic Klebsiella pneumoniae KKWB-5 of Banana |
| XIA Qi-yu1, SUN Jian-bo1, GU Wen-liang2, LU Juan2, LU Xue-hua1, WANG Yu-guang1, ZHANG Xin3 |
1. Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101,China;
2. College of Agriculture, Hainan University, Hai Kou 570228,China;
3. Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Dan Zhou 571737,China |
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Abstract 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.
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Received: 15 November 2010
Published: 26 April 2011
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