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Isolamento e caracterização do gene CaLTP EM Coffea spp

Guitton Cotta M.. 2012. Lavras : UFLA, 161 p.. Mémoire de master -- Biotecnologia Vegetal.

The non-specific lipid transfer proteins (nsLTPs) are basic proteins characterized by conserved cysteine residues, low molecular mass and high content of ?-helices that were originally defined according to their ability to transfer phospholipids in vitro. Several studies indicate that nsLTPs are involved in protection mechanisms of plants against biotic stresses caused by fungi, virus and bacteria. The lipid transfer proteins are widely distributed in plant kingdom and evolutionary evidences suggest that nsLTP proteins emerged very early during land plant evolution, which may explain the large diversity of their corresponding genes. The differential expression patterns according to tissue, developmental stage and physiological condition are conferred by nsLTPmultigene family. These genes are also responsive to abiotic stresses such as drought, low temperature, salt treatment and heavy metals exposure. Considering the importance of nsLTPs in plant metabolism, the present study aims to (i) identify the different coffee nsLTPs gene homeologs corresponding to Coffea arabica (Ca) ancestor subgenomes: Coffea canephora (Cc) and Coffea eugenioides (Ce); (ii) evaluate the expression of these alleles during bean development; (iii) study the effects of drought on nsLTP expression in C. arabica and (iv) clone the promoter of one of genes enconding C. arabica nsLTP and to evaluate its function in transgenic tobacco plants. Electronic Northern were performed using electronic data from the Brazilian Genome Project EST and identified the CaLTP (Coffea arabica Lipid Transfer Protein) gene as highly expressed in coffee fruits. Results of these in silico analyses were validated by RT-qPCR and Northern blot assays which highlighted the preferential expression in C. arabica fruit endosperm at 90 and 120 days after flowering (DAF). The corresponding CaLTP3 gene promoter region (1,2 kb) was also isolated. 5' end deletions of this sequence were carried out to evaluate their ability to control the expression of the uidA reporter gene in transgenic Nicotiana tabacum plants. The GUS histochemical assay showed that 1,2 kb, 1,0 kb and 0,8 kb fragments direct the uidA expression to all tested plant organs. Those three fragments (1,2 kb, 1,0 kb and 0,8 kb) promote low or null expression in roots. On the other hand, the 0,4 kb fragment led to the expression of the uidA gene only in seeds. The expression of CaLTP-specific homeologus from Cc and Ce subgenomes of Ca was also identified using several specific primers combination. The cDNAs and genes encoding the nsLTP type 2 protein were cloned and sequenced that allowed the identification of CaLTP homeologous and orthologous sequences (CaLTP1a, CaLTP1b, CaLTP2, CaLTP3a, CaLTP3b e CcLTP3). (Résumé d'auteur)

Mots-clés : coffea arabica; coffea canephora; coffea eugenioides; résistance à la sécheresse; génie génétique; nicotiana tabacum; plante transgénique; expression des gènes; gène homéotique; gène; séquence nucléotidique; clonage; brésil; promoteur

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