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 - 3.14

VECTOR FAMILY FOR EFFICIENT TARGETING OF FOREIGN GENES TO TOMATO CHLOROPLAST DNA

F. VANTINI*, C. GOVONI*, L. CATTIVELLI**, M. GASTALDELLI*, G. TACCONI**, R. BASSI***

*) Laboratorio di Fotosintesi, Dipartimento Scientifico Tecnologico, Università di Verona

**) Istituto Sperimentale per la Cerealicoltura, Fiorenzuola d’Arda

***) Universitè Aix-Marsille II, LGPB, Facultè des Sciences de Luminy

chloroplast, vector, transformation, tomato

Genetic information in plants is localised in three subcellular organelles: nucleus, mitochondria and plastids. For many years, biotechnological strategies were focalised on nuclear genome transformation. More recently, plastid genome (cpDNA) has become amenable to genetic engineering. Indeed, high number of chloroplasts per cell and their elevated ploidy coupled with the absence of gene silencing and position effect, get to reach unexpected high expression level of transgene. Moreover, prokaryotic-like genes organisation allows multiple genes expression with single transformation event, and maternal inheritance in most agronomical species makes plastid transformation technology environmental safe.

The production of recombinant proteins in plants has many potential advantages for generating biopharmaceuticals relevant to clinical medicine. One attractive advantage offered by the high level of foreign protein expression concerns the employment of plant edible organs in direct oral administration of active principles, e.g. edible vaccines. Therefore, we chose to induce plastid transgene expression in tomato (Lycopersicon esculentum) berries, where chloroplasts accumulate in green fruit and evolve to cromoplast during ripening.

We developed a family of tomato chloroplast integration vectors (pZAI). Expression cassettes contain multiple cloning site (MCS) at 5’ position and, downstream, two markers conferring resistance to spectinomycin (aadA) and betaine aldehyde (bet B). The operon is expressed from the 5’-(P) and 3’-(T) regulatory regions of plastidic genes: Prrn, the strong promoter of rRNA (rrn) plastidic operon and TpsbA, the 3’ region of psbA. Every expression cassette is surrounded by two flanking regions (left, FR_L and right, FR_R) that drive the insertion via cpDNA homologue recombination. In order to design specific FR, we sequenced a 5 Kb region of tomato cpDNA inverted repeats and we cloned FR in pZAI vectors (respectively 0.8 Kb and 1 Kb) that localise the insertion between trnI and trnA genes.

Plasmids were synthesised using Fusion PCR technique that allows to join all the cassette’s elements saving time in cloning. Finally, markers were tested in E. coli, because of Prrn works as well in plastids as in prokaryotes. Bacteria were plated on LB + Spect²⁰⁰ medium and BADH activity was colorimetrically measured with NAD⁺ reduction assay. In both experiments transformed E. coli proved to express active marker proteins.