Journal of Food, Agriculture and Environment

Construction of a GFP-labeled plasmid suitable for in situ environmental studies

Author(s):

Qing Yu, Anzhou Ma, Xuemei Deng, Kuo Tang, Junmei Wu, Guoqiang Zhuang *

Recieved Date: 2013-06-22, Accepted Date: 2013-09-10

Abstract:

Aerial plant leaves harbor hundreds of species of microbes, known as epiphytes which have important effects on the health of both plants and people who consume fresh fruits and vegetables. Therefore, it is necessary to collect detailed information on the nature and distribution of these microbes. In order to indicate these microbes in situ, an indicator plasmid pROBENGR was constructed based on a broad-host-range replicon plasmid vector derived from pBBR1 from Bordetella bronchiseptica. The relatively low-copy-number plasmid pROBENGR contains three important motifs, the stable motif composed by replication (rep) and mobilization (mob) units, the selection marker and the indicator gene expression cassette which can express green fluorescent protein (GFP) constitutively in host cells. After transformed into host cells, plasmid retention and expression results showed that the plasmid can be maintained mostly in wild-type strains of bacteria and can express GFP constitutively. Furthermore, the plasmid was used to the indicator experiments. When the plasmid was transformed to the wild-type strain Pseudomonas stutzeri sp. Nov. Y2011 from tobacco phyllosphere, it was sufficiently stable to indicate the labeled strain for approximately two weeks without antibiotic selection on the phyllosphere. Using the confocal laser scanning microscope, the distribution of P. stutzeri sp. Nov. Y2011 marked with plasmid pROBENGR can be well tracked on its in situ environment, the tobacco phyllosphere. Together, all these results demonstrated that this plasmid is a suitable tool for constructing wholecell indicators to study the behaviors of microbes in an in situ environment. Importantly, it provides a selective and sensitive method for the studies of food security and plant protections.

Keywords:

Broad-host-range replicon plasmid, in situ studies, phyllosphere, microbes distribution, plasmid construction

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Journal: Journal of Food, Agriculture and Environment
Year: 2013
Volume: 11
Issue: 3&4
Category: Environment
Pages: 2493-2497

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