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 - 5.14

STUDY OF THE EXPRESSION OF A CHITINASE FROM AUTOGRAPHA CALIFORNICA MULTIPLE NUCLEAR POLYEDROSIS VIRUS IN TOBACCO PLANTS

S. ARCIELLO, M. LABRIOLA, R. RAO

Dipartimento di Scienze del Suolo, della Pianta e dell’Ambiente, Università degli Studi di Napoli “Federico II”, Via Università 100, Portici

Virus gene, protoplast transformation, chitinolytic activity

Biotic stresses represent the main cause of loss of production in agriculture. Pest control actually is achieved through chemical pesticides causing problems of toxicity and environmental safety. The genetic engineering of plants, powerful tool to tranfer resistance genes to crops, can overcome limits connected with strategies actually used to protect plants.

Much attention has been addressed to chitinases for their specific hydrolytic activity towards chitin a structural homopolymer which has been found in the cell wall of fungi and in the peritrophic membrane and the exoscheleton of insects. The chitinase encoding gene has been expressed in E. coli cells and the purified protein proved to inhibit Botrytis cinerea and Alternaria alternata spore germination and to perforate peritrophic matrix of Bombyx mori larvae in in vitro assay. The recombinant enzyme showed both endo and exo chitinase activities as in the virus infected cells. With the aim to find out the best condition to express the ChiA protein in plant, Nicotiana tabacum has been stably transformed with two different contructs: the wild type chiA gene harboring a carboxy-terminal ER retention motif (KDEL) and a mutated gene missing the KDEL which was replaced with the YGAL sequence.

ChiA production in transgenic tobacco plants transformed with the two contructs was assessed by immunoblotting which revealed that KDEL substitution caused a reduction in protein accumulation. Moreover both native and mutated ChiA protein exherted enzymatic activity.

Localization studies of the recombinant proteins carryed out on protoplasts and media immunoblotting, demonstrated that wild type ChiA accumulated mostly inside the cells while mutated protein was preferentially secreted into the media. Chitinolytic assay performed on secreted proteins showed that ChiAYGAL stored its enzimatic activity proving that despite protein production was reduced, KDEL substitution did not compromise protein functionality. We expect that the different localization of the recombinant protein may be useful to protect the plants against pathogenic fungi (secreted protein), and phytophagous insect (retained protein).