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Molecular Plant Breeding 2011, Vol.2, No.8, 48
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52
1.3 Stability of transgene expression in crossing
transmission
During the course of crossbreeding for generating
different transgenic hybrid rice lines, all the hybrids
were subject to Basta-resistance assay and only the
resistant plants were selected. These ensured the
existence and expression of selected bar gene in all
hybrids. What about the fate of the non-selected
cecropin B
gene? The completeness of
cecropin B
gene expression cassette in hybrid rice lines was
examined by Southern blotting analyses and the
expression status of
cecropin B
in crossing
transmission was revealed by Northern blotting
analyses.
To detect the completeness of
cecropin B
gene
expression cassette in rice genome, Southern blot was
conducted after genomic DNA was digested with
Pst
Ⅰ
and
Hin
d
Ⅲ
, which released a 1.12 kb fragment
comprising of the coding region of
cecropin B
gene
and its pin terminator (Figure 3). Results showed that
presence of the predicted 1.12 kb fragment in hybrid
lines mainly depended on their original transgenic
donors (Figure 1C and 2C). Transgene donor TR 5,
TR 6 and their derived hybrids did not exhibit the
expected 1.12 kb fragment, illustrating there was no
intact
cecropin B
copies, or more probably, the cut
sites of restriction enzymes (
Pst
Ⅰ
and
Hin
d
Ⅲ
) in
cecropin B
gene expression cassette were modified.
Transgene donor Ming B, Jingyin 119 and their
hybrids generated the 1.12 kb fragment as expected,
revealing that there was at least one intact copy of
cecropin B
gene in these transgenic lines.
Northern blot results showed the expression behaviour
of non-selected
cecropin B
gene varied significantly
among transgenic donors and their hybrid lines
(Figure 1D and Figure 2D). The expression of
cecropin B
gene in the primary transformants (T0
generation) of all the four transgene donors including
TR 5, Ming B, TR 6 and Jingyin 119 was proved by
Northern blot analysis (data not shown). However, in
their self-pollinated offspring, gene silence of
cecrropin B
occurred in TR 5 and Ming B donor
(Figure 1D), both harbouring 3 hybridization bands of
cecropin B (Figure 1B). The TR 6 and Jingyin 119
donors, with 5 and 2 hybridization bands of
cecropin
B
gene respectively (Figure 1B and 2B), expressed
cecropin B
gene stably arcoss 6 and 12 generations,
respectively. This indicated that silencing of
non-selected
cecropin B
over generations was relevant
to different transformation events rather than the
number of integrated hybridization bands. In view of
the Southern blot results, we also concluded that there
was no certain relationship between the
cecropin B
gene expression status and the emergence of the
expected 1.12 kb fragment, which was used to predict
the completeness of
cecropin B
gene copy.
The expression behaviour of
cecropin B
gene in cross
transmission was revealed by comparing the Northern
blot results of transgenic hybrids with that of their
corresponding donors. Gene silence of
cecropin B
occurred in both self-pollinated progeny of TR 5
donor (T5) and all its derived hybrid lines (F
3
) (Figure
1D). In TR 6 transgene donor,
cecropin B
gene was
expressed at mRNA level over 6 generations, but
silenced in its two hybrid lines (F
3
generation).
Interestingly,
cecropin B
gene did not express in the
selfed progenies (T6) of transgene donor Ming B, but
expressed in its two out of three cross lines Ming
B/Jia 59 and Ming B/Xuzao, till F3 generation (Figure
1D). The stable expression of
cecropin B
gene was
observed in Jinyin 119 donor and all its hybrids,
ignoring the complex cross combinations and multiple
cross turns. We analysed the
cecropin B
expression in
the progeny plants of 14 out of 17 hybrids from
Jingyin 119 donor (Figure 2D). Whether transgnene
donor Jinyin 119 was female parent or male parent
(C20 / Jingyin 119), whether transgenes in Jingyin 119
were transferred through one cross turn (Jingyin
119/57, Jingyin 119/Bing 94-02, Jingyin 119/59), two
cross turns (Jingyin 119/59//L97-55, Jingyin 119/57//
9522, Jingyin 119/390//S1, Jingyin 119/63//T951,
Jingyin 119/Bing 94-02//T951, Jingyin 119/02//T951,
Jingyin 119/63//390, Jingyin 119/59//DS4 and Jingyin
119/503//T951) or three cross turns (Jingyin 119/ 57 //
DS4///L97-55), or stacked by crossing between
transgenic hybrids (Jingyin 119/59//DS4///Jingyin
119/31//9522), the non-selected
cecropin B
gene
expressed stably in all hybrids over 6 to 8 generations.
In general, the expression behaviour of non-selected
cecropin B
gene was complex in crossbreeding