Antje Dietz-Pfeilstetter, Nicola Arndt, Ulrike Manske
Effects of a petunia scaffold/matrix attachment region on copy-number dependency and stability of transgene expression in Nicotiana tabacum
Julius Kühn-Institut, Federal Research Centre for Cultivated Plants, Institute for Biosafety in Plant Biotechnology, Messeweg 11-12, D-38104 Braunschweig, Germany
e-mail: antje.dietz@jki.bund.de
Supplementary Fig. 1 Southern blot analysis of a representative number of LX222GUS Nicotiana tabacum transformants. (a) T-DNA of plasmid
pLX222GUS used for plant transformation and location of probe 1 (nos5´-nptII) and probe 2 (gus), (b) HindIII digested DNA hybridized to probe 1 for copy
number determination, (c) HindIII digested genomic DNA hybridized to probe 2 to control the intactness of the gus gene cassette. Pl, plasmid control. The
sizes of the expected hybridizing plasmid fragments are indicated. Due to cross hybridization of probe 1 with the internal 5.7 kb HindIII fragment an additional
band was present in all lanes.
Supplementary Fig. 2 Southern blot analysis of a representative number of BI22SAR2E Nicotiana tabacum transformants. (a) T-DNA of plasmid
pBI22SAR2E used for plant transformation and location of probe 1 (nos5´-nptII) and probe 2 (gus), (b) HindIII digested DNA hybridized to probe 1 for
copy number determination, (c) HindIII digested genomic DNA hybridized to probe 2 to control the intactness of the gus gene cassette. Pl, plasmid
control. The sizes of the expected hybridizing plasmid fragments are indicated.
Supplementary Table 1
DNA methylation pattern within the gus coding region in GUS expressing and nonexpressing BI22SAR2E-7b plants grown under greenhouse conditions. For each plant the % C-methylation resulting
from sequence analyses of six clones and the average % C-methylation are shown. Copy number and integration
structure is given for comparison
F1 plant
GUS expression
Tissue culture
Greenhouse
Integration pattern
Copy no.
Repeats
BC1-1
2.6
4.0
3
Direct tandem
RB inverted
BC1-2
0
0.9
2
No
BC1-3
0
1.3
2
No
BC1-4
0
3.2
5
Direct tandem
RB inverted
% C-methylation within 5´ part of gus gene (737 bp)
CG
CNG
CNN
44
2
0
75
29
7
42
4
4
Ø 54
Ø 23
Ø5
39
4
2
44
2
0
81
94
16
86
60
27
90
71
10
95
54
10
Ø 90
Ø 61
Ø 22
77
8
3
97
88
37
95
85
43
82
94
25
89
60
32
96
85
26
Ø 86
Ø 75
Ø 23
82
94
25
86
38
5
82
81
27
58
48
12
75
6
0
95
94
27
Ø 85
Ø 67
Ø 20
97
75
27
97
94
38
89
88
16