Basic HTML Version
Molecular Plant Breeding Provisional Publishing
Molecular Plant Breeding 2012, Vol.3, No.
6
, 57
-
62
http://mpb.sophiapublisher.com
57
Digestion of purified pG-gB+ with
Bam
H
Ⅰ
,
Nco
Ⅰ
and
Sac
Ⅰ
released a1.3 kb fragment and a 2.2 kb
fragment, respectively, as shown in Figure 1B. The
same was done with pG-gB-, and the result is shown
in figure 1C. All fragments were the same as expected.
1.2
glg
B expression in recombinant strains
To ensure
glg
B with three mutation sites function in
glycogen biosynthesis as expected, functional
confirmation of the
glg
B isolates was done by
expressing
glg
B in
E.coli
. To do so, the
glg
B was
subcloned into downstream of T7 promoter in
pET
-
28c in both 5'
-
3' and 3'
-
5' directions. Purified
recombined plasmids pE-
g
B+ and pE-
g
B
-
were
digested with
Xba
Ⅰ
and
Pst
Ⅰ
as well as
Bam
H
Ⅰ
.
As shown in Figure 1D and 1E, expected fragments
were released from pE-
g
B+ and pE-
g
B
-
.
It indicated
that the recombined bacterial strains BL-E
g
B+ and
BL-E
g
B-, containing the pE-
g
B+ and pE-
g
B
-
respectively, were constructed.
To find effect of
glg
B expression on glycogen in the
strains, glycogens from BL-E
g
B+, BL-E
g
B- and
BL
-
21 were extracted and scanned with 190-1900nm
wavelength coverage. The result was shown in Figure
2A, 2B and 2C. The absorption peaks of glycogen
from BL-E
g
B+ were the same as that from BL
-
21,
but different from those of BL-E
g
B-. An extra
absorption peak appeared between 300nm and 400nm
in glycogen from BL-EgB-, which is absent in other
strains. This indicated that the expression of reverse
glg
B driven by T7 promoter changed the glycogen
structure in BL-EgB-. Therefore, it is concluded that
glg
B isolate is functional in glycogen biosynthesis.
Figure 2 Light-absorption features of glycogen from the recombined bacteria BL-
g
B
＋
and BL-
g
B-
Note: A: BL-
g
B
＋
; B: BL-
g
B-; C: BL21
1.3
glg
B expression in potato transgenic lines
To express
glg
B in potatoes, transformation vector
pC-SaS was constructed and transformed into
agrobacterial LBA4404 to generate recombinant
strains LBA-gB. The recombined pC-gB was
extracted from LBA-gB, and it was cut with
Bam
H
Ⅰ
and
Eco
R
Ⅰ
. A 1.2 kb and a 940 bp as well as a 2.2 kb
fragment were released, respectively, as shown in
Figure 3.
26 PCR positive transformants were found from two
groups of transgenic lines. Some of those were shown
in figure 4A. Southern blot showed that among the 26
PCR positive transformants, 9 were inserted with
Figure 3 Digestion result of vector pC-gB
Note: M1.DNA molecular standard; 1: Products of
Bam
H
Ⅰ
digestion on pC-gB; 2: Products of
Eco
R
Ⅰ
digestion on
pC-gB; 3: Recombined plasmid pC-gB; M2: DNA molecular
standard