Plant Cell, Tissue and Organ Culture 52: 183–187, 1998.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
183
Research note
Modifying transient β-glucuronidase expression in pine species using
introns
Jaime M. Humara, Mari
´
an L
´
opez & Ricardo J. Ord
´
as
∗
University of Oviedo, Dept. B.O.S., Unidad Fisiolog
´
ia Vegetal, C/ Catedr´atico Rodrigo Ur
´
ia s/n, E-33071 Oviedo,
Asturias, Spain; Instituto Universitario de Biotecnolog
´
ia de Asturias-CNB (CSIC), E-33071 Oviedo, Asturias,
Spain (
∗
requests for offprints)
Received 7 July 1997; accepted in revised form 12 January 1998
Key words: uidA expression, microparticle bombardment, Pinus nigra, Pinus pinea, transient gene expression
Abstract
The effect of introns on gene expression was evaluated. Several intron-promoter combinations were introduced
by microparticle bombardment into two pine species, stone pine (Pinus pinea L.) and salgareño pine (Pinus nigra
Arn. ssp. Salzmannii (Dunal) Franco). Gene expression was evaluated by measuring transient GUS expression.
Two promoters (CaMV35S and double CaMV35S modified) and two introns (intron 1 from maize genes alcohol
dehydrogenase-1 and Shrunken-1) were used in our study. In both pine species tested, the Sh1-int1 increased
transient GUS expression from 2 to 6-fold compared to the intron-less construction. On the contrary, the inclusion
of the Adh1-int1 associated with the double CaMV35S modified resulted in a dramatic decrease in the expression
in both pine species analyzed. Our results suggest that Sh1-int1 may be useful for the acquisition of the required
levels of genetic activity of new agronomic traits introduced into pines.
Abbreviations:CaMV35S – promoter 35S of the Cauliflower Mosaic Virus; uidA –geneforβ-glucuronidase;GUS
– β-glucuronidase
Plant transformation with foreign DNA has become
a powerful tool to study mechanisms of gene expres-
sion. Enhancement of gene expression in plants by the
use of introns was first demonstrated by Callis et al.
(1987). Such enhancement requires that the intron is
present in the transcriptional unit of the gene (Callis et
al., 1987; Mascarenhas et al., 1990, Maas et al., 1991),
and it is most likely based on post-transcriptional
mechanisms (Maas et al., 1991). Research on intron
enhancement of gene expression is valuable for basic
studies on RNA processing and stability. The use of
intronsto increase heterologousgeneexpression could
be important to achieve the required levels of genetic
activity and to develop reliable, high level expression
vectors for transformation of conifers.
Intron-enhancement of gene expression has been
reported in various monocotyledonous species using
certain introns, but the situation in dicot species is
less clear. In dicot species, monocot introns do not
stimulate and often reduce gene expression, while the
effects of dicot introns vary from no stimulation of
gene expression to a slight stimulatory effect (Vain et
al., 1996).
Goodall and Filipowicz (1991) found that require-
ments for intron recognition in plants are different
fromthose of both metazoaand yeastand more similar
to the vertebrates, concluding that AU-rich sequences
are necessary for intron recognition. Moreover, they
also observed that there is a minimum functional
intronlength in both monocots and dicots, and theyin-
dicated that monocots differ substantially from dicots
in their mechanism of intron recognition, suggesting
that the splicing in monocotsis more ‘permissive’than
the splicing in dicot plants or vertebrates.
As a new step towards the successful stable trans-
formation of pines, and to investigate whether the
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