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 - 1.01

 

CHARACTERIZATION OF THE NF-Y SUBUNITS IN ARABIDOPSIS THALIANA

 

V. CALVENZANI, M. LAPILLA, T. SALA, K. PETRONI, C. TONELLI

 

Università degli Studi di Milano, Dipartimento di Scienze Biomolecolari e Biotecnologie, Via Celoria 26, 20133 Milano

 

 

transcription factor, CAAT binding proteins, NF-Y, Arabidopsis

 

NF-Y is a CCAAT-specific binding factor composed of three dinstinct subunit. In vertebrates and fungi all three subunit are encoded by evolutionary conserved single copy genes. In a statistical analysis of over 500 eukariotic promoters, the CCAAT box was one of the most ubiquitous elements, being present in 30% of them (1).

 

Most of the information about the CCAAT binding protein is provided from studies in yeast and mammals, but our knowledge of the biology of NF-Y genes in plants is still rudimentary.

 

In our laboratory all the members of the NF-Y family in Arabidopsis thaliana have been cloned. This family is composed of 29 subunits genes: 10 NF-YAs, 10 NF-YBs, 9 NF-YCs. Gene specific probes were used for the preliminary characterization of the different members by semi-quantitative RT-PCR on RNA samples obtained from several tissues and developmental stages. In general, the emerging picture is extremely complex, with all genes expressed at least in one stage of flower development and a maximum of 20 genes induced in senescent flowers, 3 days after fertilisation. The phylogenetic analysis showed the existence of different subgroups of sequences inside each family, with a good correlation between the belonging to a certain subgroup and the corresponding expression patterns (2, 3).

 

In order to identify the NF-Y subunits of Arabidopsis which interact with other transcription factors, 25 out of 29 Two-Hybrid  constructs have been prepared. In particular, the complete ORF of 24 subunits (7 NF-YA, 8 NF-YB e 9 NF-YC) has been fused in frame to the activation domain of Gal4 (AD vectors) or to the DNA binding domain of Gal4 (BD vectors). The constructs have been sequenced to verify the frame and have been introduced in yeast to check them for autoactivation. The constructs prepared were used to test, in a Two-Hybrid screening, the possible interactions with different transcription factors from Arabidopsis prepared together with other laboratories within an European project. The positive results will be confirmed and these genes will be functionally characterized by searching of insertional mutants in Arabidopsis T-DNA and transposon populations. Moreover the phenotypes of the double mutants between genes  belonging to the same subgroup will be further characterised.

 

 

(1) Mantovani (1998), Nucleic Acid Res. 26:1135-1143

(2) Gusmaroli et al. (2001), Gene 264:173-185

(3) Gusmaroli et al. (2002), Gene 283:41-48