Page 7 - Molecular Plant Breeding

Molecular Plant Breeding 2011, Vol.2, No.8, 48 -59
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Figure1 Integration and expression analyses of selected bar and
non-selected cecropin B gene in rice in mono crossing transmission
Note: A: Southern blot of bar gene: genomic DNA was
digested with Hind III, which cut once in the plasmid pCB1 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 Hind Ⅲ 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 Hind Ⅲ and PstⅠ 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 markerIII (Roche); Lane P: Plasmid pCB1
control; Lane U: Untransformed rice plant control; Lane T:
Jin-yin 119 transgenic rice plant positive control; Lane 1: TR 5
transgene donor, Lane 2: TR 5/CJN 3; Lane 3: TR 5/CJ 601;
Lane 4: TR 5/CJ 683; Lane 5: TR 5/Bing 97-267; Lane 6: Ming
B transgene donor; Lane 7: Ming B/Jia 59; Lane 8: Ming B/Jia
60; Lane 9: Ming B/Xuzao; Lane 10: TR 6 transgene donor;
Lane 11: TR 6/CJN 2; Lane 12: TR 6/Bing 95-13; Lane 13: TR
5/TR 6
same as that of their corresponding transgene donors
(Figure 1B). However, the integration patterns of bar
gene were changed in some hybrids (Figure 1A). For
example, two bands of 1.5 kb and 2.0 kb in length
were lost from the progeny plants of cross lines TR
6/CJN 2 and TR 6/Bing 95- 13, comparing with their
transgene donor TR 6. Two new hybridization bands
of bar gene, 1.5 kb and 2.0 kb in length, emerged in
the progeny plant of cross line TR5/CJN 3, comparing
with its transgene donor TR5. Although the TR5 lane
was loaded with less DNA, giving possibility of the
1.5 kb and 2.0 kb bands of bar gene coming from its
parent TR5, these two new hybridiztion bands did not
appear in the remaining other three hybrids from TR5,
whoes lanes were loaded with more DNA (Figure 1A).
These confirmed the conclusion that the 1.5 kb and
2.0 kb hybridization bands of bar gene were created in
cross line TR5/CJN 3.
1.2 Stability of transgene integration patterns in
multiple crosses transmission
The inheritance of transgene bar and cecropin B in the
course of multiple crosses was revealed by DNA
Southern blotting analysis, using Jingyin 119 line as
transgene donor. This Jingyin 119 transgenic line had
four bar gene loci and two cecropin B gene loci when
hybridized with their probe respectively, after
genomic DNA was digested with Hind Ⅲ, which cut
once in the plasmid pCB1 (Figure 2A and 2B).
Southern blotting results demonstrated that the
integration pattern of non-selected cecropin B gene
was very stable in mono- and multiple crossbreeding
transmission (Figure 2B). All the progenies of hybrids
showed two hybridization bands of cecropin B gene,
exactly the same as that of Jingyin 119 donor. But the
integration pattern of selected bar gene did not always
remain stable during crossbreeding transmission.
Among the seventeen rice crosses, the integration
pattern of bar gene remained stable in eight hybrids
and changed in the other nine lines, which lost two
smaller hybridization bands of bar gene, 1.6 kb and
1.0 kb in length respectively (Figure 2A). Our former
research revealed that the transgenic integration
patterns of Jingyin 119 transgene donor kept stable in
self-pollination across generations, but bar and
cecropin B gene showed different integration patterns