Construction
of a vector for the Phoc gene stable chromosomal
integration in Plant
Growth Promoting Rhizobacteria (PGPR)
Cuban Research
Institute on SugarCane by-Products (ICIDCA), Dept. of Microbiology, P.O. Box
4026 CH-10 CP: 11000, Havana. CUBA
E-mail: rey@icidca.edu.cu
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Abstract
Acid phosphatases play a major role in the mineralization of organic phosphorous in soil, so genetic manipulation by means of recombinant DNA technology seems to offer feasible approaches for obtaining improved phosphate solubilizing strains. A plasmid was constructed using the suicide delivery vector pJMT6, a pUT/mini Tn5 derivative vector and the plasmid pLR1, the latter one harbouring a gene encoding the PhoC acid phosphatase form Morganella morganii. The recombinant construction pLF17, which contains a non-antibiotic resistance selection marker (resistance to potassium tellurite), was transformed and expressed in Escherichia coli CC118lpir. A transformant clone, E. coli CC118lpir F17 (harbouring pLF17) was selected and further characterized, showing the phoC gene expression through a histochemical assay based on an indicator medium and zymograms developed to detect phosphatase activity, with this technique it was possible to detect in the whole cell extracts and in the supernatant fraction the polypeptidic component of 25 kDa which was the responsable of the acid phosphatase activity. The acid phosphatase activity was quantified in the whole cell and in the supernatant of the culture being higher in the transformant E. coli CC118lpir F17 than E.coli CC118lpir without plasmid at all times analyzed. The objective of this work was the subcloning of the gene phoC in an appropriate vector that will permit stable chromosomal integration of this gene in plant growth promoting rhizobacteria (PGPR) in order to avoid the horizontal transfer of the inserted gene in soil and prevent the risk of metabolic load caused by the presence of the plasmid in the cell.