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
PRELIMINARY STUDIES FOR OBTAINING FUNCTIONAL MARKERS IN TUBULIN GENES
OF BARLEY
VIGNANI R., SENSI E., SCALI M., CRESTI M.
Dipartimento di Scienze Ambientali, Università
degli Studi di Siena
scalimoni@unisi.it
tubulin, cytoskeleton, functional markers, Hordeum vulgare
Cytoskeleton is an
ubiquitous structure which participates to multiple crucial developmental
processes in plant and animal cells. Microtubules whose protofilaments are
heterodimers of a and b tubulin, are among
the structural components of the cytoskeleton and in plant cells they are
involved in many processes such as cell division, intracellular transport and
control of cell shape. Cortical microtubules array forms a complex dynamic
structure which is supposed to take a significative role in the regulation of
the intracellular streaming occurring in growing pollen tube (1). The pollen
tube is a suitable and convenient model for the study of plant cytoskeleton
functionality and regulation (2) since it exhibits such a high speed of
space-directional elongation which is promoted by the intracellular streaming.
The complex morphological dynamism which is observed in the pollen tube
cytoskeleton makes this type of cell a good system for investigating the
putative tubulin isotype specialization during a crucial developmental phase of
higher plants such as the pollen tube emission and growth.
Plant tubulin genes
have been characterized in many different species (3, 4), but the pattern of
expression of each either a or b characterized gene
is best known for Arabidopsis thaliana (5, 6, 7) and Zea
mais (8).
The development of
assay for functionally relevant regions of the genome is a recent aim of the
scientific community involved in biodiversity conservation.
A set of different
barley cultivars has been screened in order to verify the stability of a
microsatellite element which is present in the upstream region of the a- tubulin 1 gene in
order to see if this region could be used as possible DNA marker related to
tubulin genes.
Current PCR based
techniques have been used to evaluate the potentiality of such region of the
genome to act as functional marker. Primers anchored in the conserved regions
on cDNA, which include the microsatellite, lead to production of a major band
of approximately 1100bp. Sequence analysis of this product revealed the
presence of an intervening sequence of 896 bp which could be interpreted as a
non-coding region (putative intron I) of the gene coding for a-tubulin1. The
putative intron in barley is positioned at aa position 38 in agreement to Arabidopsis
intron I position of TUA5, 3 and 1. Sequences present in
GeneBank coding for tubulins in Hordeum vulgare include six
different accessions for a-isoforms
coding genes, three for b-tubulins
and one for g-tubulin.
None of the sequence available for this species reports a description of
intervening sequences.
Preliminary
sequence alignment of 1100 bp PCR products obtained for 3 different barley
cultivars shows that they are almost identical except for a few point mutations
in the intron.
Further analysis of
the intervening sequence isolated show the presence of several consensus motifs
and of generic splicing sites. The GT-AT rule is followed and a branch
consensus sequence is located at –31 nt (TATTAAT). The putative intron
region shows an AT content which reaches 58% against 36-38% with respect to
related coding parts of the same gene.
(1)
Cai et
al. 1997, Trends in Plant Sci 2: 86-91
(2)
Moscatelli et al. 1995, J Cell Sci 108: 1117-1125
(3)
Fosket 1989, In the biochemistry of plants, Vol. 15 PK
Stumpf ed., New York, Academic Press pp. 392-454
(4)
Hussey et al. 1991, The cytoskeletal basis of plant
growth and form, C.W. Lloyd ed., London, Academic Press
(5)
Kopczac et al. 1992, Plant Cell 4, 539-547;
(6)
Snustad 1992, Plant Cell 4: 549-556;
(7)
Chu et al 1998, Plant Mol Biol 37: 785-790
(8)
Villemur et al.,1994 Plant Mol Biol 24: 295-315