Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 3.16

IN PLANTA EXPRESSION OF A FUNGAL LACCASE GENE USEFUL FOR PHYTOREMEDIATION OF PAHs CONTAMINATED SOILS

GALANTE C., ESPOSITO S., STILE M.R., MONTI L., FILIPPONE E.

Department of Soil, Plant and Environmental Sciences, School of Biotechnology, University of Naples “Federico II”, Via Università 100, 80055 Portici (Italy)

cgalante@unina.it, filippon@unina.it

laccase, poxC, phytoremediaton, PAHs, transgenic plants

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed and relocated in the environment as a result of the incomplete combustion of organic matter. Many PAHs are highly toxic, mutagenic and/or carcinogenic to microorganism as well as to higher organisms, including humans. Plants have been claimed to have the capability to produce both degradation enzyme in root exudates and a better environment for the growth of useful microbia in the rhizosphere, allowing them to exert their degradation potentiality. To enhance plants PAHs degradation ability, our research approach was to express in planta fungal phenol-oxidases (laccases), which, released by means of exudates roots, degrade condensed PAHs into less toxic compounds. The poxC laccase-encoding gene¹, from the white-rot-fungus Pleurotus ostreatus, was used in our experiment. In silico analysis of POXC peptide sequence predicted the extracellular localization of this enzyme in plant. The cDNA of poxC gene was inserted into the pKYLX binary vector, under the CaMV35S2 constitutive promoter, then the vector was transferred into Agrobacterium tumefaciens LBA4404 strain and used for agro-infiltration and for stable genetic transformation experiments of Nicotiana tabacum cv Samsun NN. The western analysis on total protein extract from agro-infiltrated leaves confirmed the presence of the transgenic enzyme. The regenerated shoots, selected in presence of kanamycine, were analysed by PCR, using specific primers for the poxC gene and for the A. tumefaciens vir genes region: all putative transgenic shoots were only positive to the amplification of poxC gene. The RT-PCR analysis confirmed the transcript presence in transgenic plants and pointed out different levels of specific transcript. Transgenic plants, growing in vitro conditions, were phenotypically different from the control untransformed ones. In particular, they were smaller than controls and they showed leaves with red-brown patches. However, roots growth and morphology were comparable with control plants. The observed differences in growth and in morphology between the organs of the transgenic plants in comparison with the untransformed ones should be related to the interference of the over-produced laccase on tobacco lignin biosynthesis. Other experiments are in course to ascertain the enzymatic activity of root exudates and to drive poxC gene expression into plant roots only.

Acknowledgements: we thank G. Sannia and P. Giardina (Dept. of Organic Chemistry and Biochemistry, University of Naples “Federico II”, Italy) for providing us the P. ostreatus poxC gene.

References

1 Giardina et al. (1999) Biochem. J. 341: 655-663