Proceedings of the XLVI Italian
Society of Agricultural Genetics - SIGA Annual Congress
Giardini
Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1
Poster
Abstract - 3.31
ISOLATION AND
CHARACTERIZATION OF cDNA SEQUENCES OF WHEAT MADS BOX GENES
PAOLACCI
A.R., ALOISIO E., CECCHETTI S., TANZARELLA O.A., PORCEDDU E., CIAFFI M.
Dipartimento di Agrobiologia e
Agrochimica, Università degli Studi della Tuscia, Via S. Camillo De
Lellis, 01100, Viterbo
wheat, flower
development, homeotic genes, differential analysis, gene expression
The genetic analysis of mutant plants of Antirrhinum and Arabidopsis defective in floral homeotic MADS-box
genes has provided evidence for a functional role of MADS-box genes in flower
development and led to theoretical models for flower morphogenesis. MADS-box
genes encode a conserved DNA-binding domain present in a variety of
transcription factors. The isolation and expression analysis of a large number
of MADS-box genes from different plant species indicated that these genes might
not only be involved in flower development, but also in vegetative
organogenesis. On the basis of phylogenetic analysis plant MADS-box genes have
been classified into 13 subfamilies. Although different MADS-box genes have
been isolated and characterized in monocot species such as maize, rice and
barley, little is known on their function in these species. Moreover, in wheat
most of the genes involved in the control of floral initiation and development
remained to be cloned and characterized, as only three MADS-box genes belonging
to the AP1, AP3 and AGL6 groups had been isolated. The aim of the
present research consists in the identification, cloning and characterization
of genes specifically expressed in wheat spikelets at different heading stages.
RNAs were collected from spikes and leaves of the bread wheat cv. Chinese
Spring and were analysed by SDDM (simple differential display method), which
consists in the reverse transcription of mRNA using random hexanucleotide and
then PCR amplification by decamers. Forty out of 60 decamers used in the
expression analysis detected a total of 65 differential PCR products between
spikes and leaves. The search of their nucleotide and translated sequences in
different genebank and EST databases indicated that eight are homologous to
different MADS-box genes. In particular, three sequences corresponded to the MADS-box
genes already isolated in wheat, two showed homologies with wheat EST
sequences, whereas the remaining three did not show any significant homology
with sequences cloned from Triticum
species. A search in HARVEST, a database containing more than 100000 EST
sequences from different Triticum
species, indicated the presence of at least nine additional sequences encoding
for wheat MADS-box transcription factors. We have amplified by RT-PCR and
cloned all the nine sequences. The size of the 17 cloned cDNA sequences ranges
from about 400 to 650 bp and all include the conserved sequence encoding for
the MADS-box domain. Based on sequence comparison to known MADS-box genes, the
wheat MADS-box (WM) cDNA sequences were assigned to 11 distinct phylogenetic
subclasses of the MADS-box gene family. The MADS-box cDNA sequences WM6, WM7
and WM8 share similarities with genes of the AP1 subgroup, WM2 and WM13 with genes of the
AG and AP3 subgroups, respectively, WM14 and WM15
with genes of the PI
subgroup, WM3 belong to the AGL11
subgroup and WM1 e WM9 cluster into the TM3 and AGL6 subgroups, respectively, whereas WM4,
WM5, WM10 and WM11 are members of the AGL2 subfamily. The remaining three MADS-box cDNA sequences
resemble MADS-box genes described as solitary sequences or orphan genes. The
short-term goals of the research will be: I) isolation of full length cDNA
sequences of some of the most interesting MADS-box genes by RACE; II)
expression analysis of the MADS-box cDNAs by Northern blots and/or RT-PCR
experiments of RNA from different plant tissues (adult leaves, coleoptile,
roots, kernels and spikes at different development stages) and from different
floral organs (glumes, lemmas, paleas, lodicules, stamens and pistils); III)
evaluation by Southern analysis of copy number per genome and chromosomal
location of the isolated full length cDNA sequences.