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Molecular Plant Breeding 2011, Vol.2, No.8, 48
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51
Figure 2 Integration and expression analyses of selected
bar
and non-
selected
cecropin B
gene in rice in multiple crossing transmission
Note: A: Southern blot of
bar
gene: genomic DNA was
digested with
Hin
dIII and hybridized with DIG-labeled
bar
probe, comprising
bar
gene coding region and
nos
teminator
(0.9 kb); B: Southern blot of
cecropin B
gene: genomic DNA
was digested with
Hin
dIII and hybridized with DIG- labeled
cecropin B
probe, comprising
cecropin B
coding region and
Pin
terminator (1.12 kb); C: Southern blot of the intact of
cecropin
B
gene: genomic DNA was digested with
Hin
d
Ⅲ
and
Pst
Ⅰ
and hybridized with
cecropin B
probe, which generated a 1.12
kb fragment as
cecropin B
probe sequence; D: Northern blot
analysis of the non-selected
cecropin B
gene expression. Lane M:
DNA molecular weight marker
Ⅲ
(Roche); Lane U:
untransformed rice plant control; Lane 1: Jingyin 119 transgene
donor; Lane 2: C20/ Jingyin 119; Lane 3: Jingyin 119/57; Lane
4: Jingyin 119/Bing 94-02; Lane 5: Jingyin 119/59; Lane 6:
Jingyin 119/104; Lane 7: Jingyin 119/59//L97-55; Lane 8:
Jingyin119/57//9522; Lane 9: Jingyin 119/390//S1; Lane 10:
Jingyin 119/63//T951; Lane 11: Jingyin 119/Bing 94-02//T951;
Lane 12: Jingyin 119/02//T951; Lane 13: Jingyin 119/63//390;
Lane 14: Jingyin 119/59//DS4; Lane 15: Jingyin 119/503//T951;
Lane 16: Jingyin 119/59//T951; Lane 17: Jingyin 119/57//DS4
///L97-55; Lane 18: Jingyin 119/59//DS4/// Jingyin 119/31//9522
when Southern-blotting analyses were conducted after
genomic DNA digested with different enzymes (Hua
et al
., 2003). During crossing transmission the two
smaller hybridization bands of
bar
gene were lost in
some hybrids while the other two bigger bands were
transmitted stably (Figure 2A and 2B).
We also noticed that disappearance of the two smaller
fragments (1.6 kb and 1.0 kb) of
bar
gene did not
depend on the cross turns (Fig. 2A). For example, the
cross hybrid of 119/Bing 94-02 kept the same
integration pattern
of
bar
gene as that of its transgene
donor Jingyin 119, but in its related re-cross hybrid
Jingyin 119/Bing 94-02//T951, the 1.6 kb and 1.0 kb
hybridization bands of
bar
gene were lost. Another
hybrid rice line of cross Jingyin 119/59 lost the two
smaller fragments of
bar
gene, but in progenies of its
four related multiple crossing hybrids, three crossing
combinations including Jingyin 119/59//L97-55,
Jingyin 119/59//DS4 and Jingyin 119/59//DS4///
Jingyin 119/31//9522 lost the two smaller fragments of
bar
gene, the remaining one cross Jingyin
119/59//T951 exhibited the same
bar
gene integration
pattern as that of the original Jingyin 119 donor. There
was also one hybrid line Jingyin 119/57//DS4///
L97-55 that carrying all the original four
bar
gene loci
of its transgene donor after three crossing turns. These
suggested that loss of
bar
gene fragments in
transmission of multiple crosses was not related to
crossing turns, but mainly depended on the primary
hybrid plants that were randomly selected as transgene
donors for the next crosses.
Moreover, disappearance of the
bar
gene smaller
fragments occurred in both hybrids when transgene
donor Jingyin 119 was male parent (C20/Jingyin 119)
and female parent (e.g. Jingyin 119/59). This
demonstrated the
bar
gene smaller fragments
harbouring in donor plant had equal chance to be lost
through pollen and egg. Therefore, we speculated that
the two smaller hybridization bands of
bar
gene in
Jingyin 119 transgene donor might integrate in one
separate genomic locus from the other bigger ones and
more possibly, possess no expression activity.