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