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

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

EXPRESSION OF THE MAIZE B32 RIP PROTEIN IN WHEAT (TRITICUM AESTIVUM L.) AND ITS IMPLICATIONS AS DEFENCE GENE AGAINST FUNGAL PATHOGENS

LUPOTTO E.*, REALI A.*, CARRARA N.*, CAFFARRI S.*, VACCINO P.**, CATTANEO M.**, FORLANI F.***

* Istituto Sperimentale per la Cerealicoltura, Sezione di Bergamo

eurice.elu@spm.it

** Sezione di S.Angelo Lodigiano (LO)

*** Dipartimento di Scienze Molecolari Agroalimentari, Università degli studi di Milano

wheat (Triticum aestivum), genetic transformation, RIP, fungal pathogens, defence genes

The maize gene b32, normally expressed in the endospem, encodes for a RIP (Ribosome Inactivating Protein). Ectopic expression of b32 in transgenic tobacco tissues, confers a certain degree of tolerance to the fungal pathogen Rhizoctonia solani AG4. The b32 protein displays a biological activity on a wide range of diseases and therefore it presents itself as a plant multipurpose defence gene. Moreover, being a naturally occurring protein in the seed protein pool of another cereal species, b32 offers an actractive rationale for its use in a biotechnological approach aimed to crop protection and consequently, to a biological control of micotoxin production.

An expression vector was constructed, in which the b32 gene is driven by the 35SCaMV promoter for its constitutive expression in all plant tissues, in association to the bar gene as a selectable marker. The genes were introduced into hexaploid wheat (Triticum aestivum L.) via microbombardment of pre-cultured immature embryos. After selection, among 170 regenerated plants, five independent events of transformation containing the b32 gene were chosen for further characterisation. T1 fully fertile plants, were analysed in Southern blot to confirm transmission of the transgene. Polyclonal antibodies anti-b32 were raised in rabbits injected with GST-b32 expressed in E.coli. Leaf protein extracts were analysed in western blots for detection of b32 expression. Plants from two wheat genotypes, Bob White and Veery, showed consistent expression of b32 in the leaf protein extracts. Moreover, the b32 protein is consistently expressed during all vegetative and reproductive stages of the plant life. An initial quantitative estimate of the wheat leaf-expressed b32 reveals that it is present at significative levels, comprised between 0.5 and 2% of the total soluble proteins.