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Molecular Plant Breeding 2011, Vol.2, No.8, 48
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promoted the plasmid rearrangements during integration,
but might exert effects on the DNA recombination
events of
bar
gene loci in the subsequent crossing
transmission. In deed, the presence of new transgenic
fragments of
bar
gene in hybrid TR 5/CJN 3 further
confirmed the rearrangement of
bar
gene integration
loci. More importantly, we found that the variance
existing in integration patterns of
bar
gene did not
interfere with its stable expression and the successful
selection of the corresponding hybrids. This indicated
that transgenes introduced by particle bombardment
could be successfully transferred in conventional
crossbreeding.
2.2 Stability of transgene expression in crossing
transmission
Different integration sites, copy numbers and
transgenic locus configurations, as well as epigenetic
silencing mechanisms are revealed to be the main
factors influencing transgene expression by previous
researchers (Iyer et al., 2000; Meyer, 1995; Matzke
and Matzke, 1998). However, documents on the
relationship between transgene expression and
crossing transmission are very limited up to date.
Several reports described the expression behaviour of
transgenes in rice crossbreeding in recent years, which
giving the opinion that at least the same transgene
expression level in hybrids, if no more than that of the
transgenic parents, could be expected (Chen et
al.,2005; Tang et al., 2006; 2007; Wang and Lin, 2007).
But the above results were established on the base of
transgenic donors containing single-copy exotic gene
and transformed by
Agrobacterium tumefaciens
method. Our results revealed the different fate of
transgenes in crossbreeding transmission that was
introduced by DNA direct delivery system.
The selected
bar
gene is expressed in all hybrids and
their corresponding transgenic parents across
generations in spite of its variability in integration
patterns. However, the co-expression behaviour of
non-selected
cecropin B
gene was very complex. Both
inactivation (e.g. in hybrids of TR 6) and maintaining
of successive expression activity (e.g. in hybrids of
Ming B) of
cecropin B
gene were observed through
crossing transmission. The stable expression of
cecropin B
gene were also observed after kinds of
cross combinations across several generations (e.g. in
hybrids of Jingyin 119 donor). We confirmed that
expression status of selected
bar
and non-selected
cecropin B
was independent within the same genomic
locations. This means the inactivation of
cecropin B
does not spread over to the adjacent
bar
gene, which
is consistent with the conclusion of some previous
reports (Vain et al., 2002; Kohli et al., 2003) but
argues against others (Lindsay et al., 1996).
As expression status of non-selected
cecropin B
gene
was very different among the four transgenic donors
in crossing transfer, we concluded that the structure of
transgenic loci and the chromosomal locations where
transgenes integrated are main factors influencing
gene expression in sexual reproduction as well as in
conventional crossbreeding. TR 5 is a typical
transgenic line that non-selected cecropin B gene
expressed in primary transformant but silenced in
progenies of both self-pollination and crosses. This
indicated the integration structure and/or transgenic
loci of
cecropin B
in TR 5 are prone to trigger gene
silence. The instability of selected
bar
gene
integration pattern in TR 5 hybrids illustrated that
transgenic loci of TR 5 line were recombination
triggering. Silencing of transgene is often aroused by
homologous sequences between multiple transgene
copies or between exotic DNA and endogenous DNA
sequences of the host plant, either the homologous
sequences at allelic or nonallelic chromosomal
locations (Kumpatla et al., 1997). It is probable that
homologous sequences of different copy or fragment
of
cecropin B
gene caused its own inactivation. The
possibility of interactions between actin promoter and
endogenous homologous sequences in rice genomic
DNA could not be ruled out, for the coding region of
cecropin B
gene is driven by actin promoter from rice.
Homology-mediated gene silencing is based on
DNA-DNA pairing, which might be involved in the
case where longer time and more generations are
needed. It could be strongly triggered when transgenes
are arranged in palindromic manner or in inverted
repeat (IR) (Fojtová et al., 2006). Whether IR exists in
transgenic loci of TR 5 needs to be revealed by
extensive research on the sequence of transgenic loci
in this line.