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Molecular Plant Breeding 2011, Vol.2, No.8, 48
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53
transmission
，
depending on its integration structures
as well as the genetic background of host plants.
1.4 Heredity and expression behavior of transgenes
in F1 hybrid of transgene donors
The heredity and expression behaviour of the exotic
bar
and
cecropin B
gene in F1 hybrid of transgene
donor TR 5 (T5 generation) and TR 6 (T6 generation)
was investigated. Southern-blot results showed that in
F1 hybrid of TR 5/TR 6,
bar
and
cecropin B
gene
exhibited the complementary integration patterns to
that of their parents (Figure 1A and 1B), suggesting
transgenic integration locations in TR 5 and TR 6
were non-allelic. But a 2.0 kb hybridization band of
bar gene from TR 6 parent was lost, further
confirming the instability transmission of selected bar
gene. As to the expression of transgenes, the F1 hybrid
rice plant exhibited Basta-resistance, implying the
normal expression of selectable
bar
gene. However,
the non-selected
cecropin B
gene was only expressed
in TR 6 parent plant and silenced both in TR 5 parent
and the F1 hybrid (Figure 1D). It seemed that the
silencing status of
cecropin B
in TR 5 could exert
negative effect on
cecropin B
gene expression in F1
hybrid.
2 Discussion
2.1 Stability of transgene integration patterns in
crossing transmission
Whether transgenes could be transferred from
transgenic donor plants into other varieties through
conventional crossbreeding is crucial for the
successful application of transgenic technique in plant
breeding. Present results described the stability of
integration patterns and expression activities of
transgenes in rice during the course of crossbreeding
transmission.
We found in process of crossing transfer, the
integration patterns of non-selected
cecropin B
gene
were very stable. All the investigated hybrids kept the
same
cecropin B
integration patterns as that of their
corresponding donor plants. However, the integration
patterns of selected
bar
gene showed instability to
certain degree. One obvious phenomenon is gene
losing. The lost hybridization bands of
bar
gene had
relatively smaller molecular weight (no more than
2.0 kb), usually companying with the stable transfer
of two or more larger Southern hybridization bands.
These indicated that the stably transferred larger bands
of
bar
gene were expressed and mainly provided the
Basta-resistance for selection of transgenic hybrids.
The smaller bands of
bar
gene that tended to be lost in
crossing transmission might integrate in different
chromosomal location from the larger ones. In the
course of crosses, these smaller bands were transferred
as unlinked (as in TR 6) or partly-linked (as in Jingyin
119) unit with the larger bands of bar gene, and most
probably possessed no expression activity, as there
was no hybrids harbouring the smaller hybridization
bands of
bar
gene only. We speculated that when these
smaller bands of bar gene happened to be caught by
hybrid plants independently, the hybrid plants could
not survive because every hybrid in our experiments
was subjected to Basta-resistance screening.
Also, there is the possibility that the loss of
bar
gene
fragments was aroused from DNA recombination
events involved in transgenic loci. Gene loss from one
generation to the next was previously reported in
maize (Register et al., 1994) and rice (Vain et al.,
2002). Some transgenic loci generated by particle
bombardment are likely to be altered from one
generation to the next through recombination or
deletion (Vain et al., 2002). Molecular studies have
shown that direct DNA transfer often leads to
integration at one locus of multiple fragmented and
rearranged transgene copies as well as plasmid
backbone sequences (Kohli et al., 2003). In the four
transgene donor plants, differences between the
integration patterns of
bar
and
cecropin B
gene were
investigated, although they were tightly linked in the
plasmid construction. These illustrated that rearrangement
or recombination might occur between
bar
and
cecropin B
gene cassettes during their integration. The
19 bp palindromic sequence within TATA-box of the
CaMV35S promoter is proved to be a recombination
hotspot that can confirm the predominance of
microhomology-mediated recombination of plasmid
rearrangement in transgenic integration (Kohli et al.,
1999).
Bar
gene in present research is driven by
CaMV35S promoter. Thus the recombination
triggering sequences in CaMV35S promoter not only