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Isolation of a strong promoter fragment from endophytic Enterobacter cloacae and verification of its promoter activity when its host strain colonizes banana plants

文献类型： 外文期刊

作者： Wang, Yu Guang 2 ; Xia, Qi Yu 2 ; Gu, Wen Liang 3 ; Sun, Jian Bo 2 ; Zhang, He 1 ; Lu, Xue Hua 2 ; Lu, Juan 3 ; Peng, Min 1 ;

作者机构： 1.Chinese Acad Trop Agr Sci, Environm & Plant Protect Inst, Danzhou 571737, Peoples R China

2.Chinese Acad Trop Agr Sci, Inst Trop Biosci & Biotechnol, Key Lab Trop Crop Biotechnol, Minist Agr, Haikou 571101, Peoples R China

3.Hainan Univ, Coll Agr, Haikou 570228, Peoples R China

关键词： Endophyte;Enterobacter cloacae;Fluorescence observation;GFP;Promoter cloning

期刊名称：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY （ 影响因子：4.813； 五年影响因子：4.697 ）

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收录情况： SCI

摘要： To engineer endophytic Enterobacter cloacae as a biocontrol agent against banana fusarium wilt, a promoter-probe plasmid pUCK was constructed to identify a strong promoter to express disease resistance genes. Using a kanamycin resistance gene for selection, 10 fragments with strong promoter activity were identified from the genome of the E. cloacae KKWB-10 strain. The regions of these 10 fragments that were the primary contributors to the promoter function were identified, and their promoter activities were further evaluated using green fluorescent protein (GFP) as a reporter gene. Fragment 132a″ drove the highest level of GFP activity when the bacteria bearing the fragments were cultured in Luria-Bertani and banana stem extract media. The GFP-expressing strain harboring fragment 132a″ (K-pUCK7-132a″-GT) was then inoculated into banana plantlets (about 1 × 10 ~7 CFU per plant) to verify the activity of fragment 132a″ in planta. Ten days after inoculation, tissue sections of these banana plantlets were observed by laser confocal scanning microscope. Green fluorescence was observed in the tissues of banana plantlets inoculated with K-pUCK7-132a″-GT but not in uninoculated controls. These results suggest that fragment 132a″ possesses strong promoter activity when its host strain colonizes the banana plants and can be used to engineer endophytic E. cloacae KKWB-10 for biocontrol.

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