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Molecular Plant Breeding Provisional Publishing
Molecular Plant Breeding 2012, Vol.3, No.
6
, 57
-
62
http://mpb.sophiapublisher.com
60
it can never be neglected that even though
glg
B is
completely inserted and expressed, alteration of starch
biosynthesis resulting from
glg
B may be different in
different genotypes of a plant, because initiation and
regulation of starch accumulation in the early stage is
variable among different genotypes. Therefore, a
further study on the transgenic lines is needed to
reveal variation in stabilized accumulated starches.
Starches used in this study were isolated from
immature tubers, which are different from the full
mature tuber; therefore, immature tubers may be
different in the starch composition from mature tubers.
Though non-transformed genotypes grown under the
same condition are regarded as the control in
comparing results of starch test in the study, further
assessment on the starch features in different
transgenic lines is needed when mature tubers are
harvested outdoor.
3 Material and Methods
3.1
glg
B isolation
Genome DNA was isolated following instruction as
described on Molecular cloning Molecular Cloning: A
Laboratory Manual (
Third Edition
) (CSH Press 2001).
According to
E. coli
JM109
glg
B DNA sequence
(AEO16768) in Genbank, primer pairs PF1 and PR1
were designed. PF1: 5'
-
cagttccatggggtgacacaat
-
3',
and PR1: 5'
-
ccctgactagtcgtaatgc
-
3'. With diluted
JM109 genome DNA and the primers, 25 μL PCR
reaction was set as 10 × PCR buffer solutions (2 μL
10 mM dNTP, 1 µL 1 μmol/L PF
1
, 1 µL 1 μmol/L PR1,
0.25 μL
pfu
DNA polymerase). Sterilized DDW was
added to fill it up to 25 µL. After mixing them, PCR
amplification was performed.
The reaction was carried out as following: denaturation
at 95
℃
for 30 sec, annealing at 52
℃
for 60 sec,
extension at 72
℃
for 3.5 min, (30 cycles in total)
extension at 72
℃
for 10 min and keeping it at 4
℃
for
the last cycle. PCR products were recovered with a gel
recovery kit (Promega).
To set up the ligation reaction of PCR products and
vector, a reaction was done with 3 µL recovered PCR
products and 1 µL pGEM-T easy vector following
instruction of the pGEM-T easy kit. 2 µL ligation
reaction products were transformed into
E. coli
supercompetent cells by heat shock. Transformed cells
were plated on a LB patri dish. Cells were grown
overnight. The white clones were screened. Positive
clones were selected by
Nco
Ⅰ
and
Sac
Ⅰ
digestion
analysis and confirmed by sequencing (ABI 3730XL
DNA Sequencer at the Shenggong, Shanghai, China).
Target recombined bacterial strains BL-gB+ and
BL-gB-, containing
glg
B in 5'
-
3' (pG-gB+) and 3'
-
5'
(pG-gB-) direction, respectively were obtained.
3.2 Construct of pE-
g
B
Isolate recombinant plasmid pG-gB+ and pE-GB. Cut
both DNA with
Nco
Ⅰ
and
Sac
Ⅰ
. Recovery and
purify cut fragments. Ligate the fragment with T4
DNA ligase and transform it into the super competent
cells of host bacteria BL21. The recombined plasmid
pE-gB+, in wh ch
glg
B was downstream of the T7
promoter was obtained. Likewise, the recombined
plasmid pE-gB- was obtained. Recombined bacterial
strains BL-E
g
B+ and BL-E
g
B-, containing pE-gB+
and pE-gB- respectively were proven by digestion
analysis with
Nco
Ⅰ
and
Sac
Ⅰ
as well as
Bam
H
Ⅰ
.
Glycogen from recombined bacterial strains was
isolated and measured in accordance with methods
used by Haugen et al (1976).
3.3 Construction of pC-gB
Isolate pC-SaS from the
Agrobacterial
LBA4404-SaS,
pG-gB from the recombined bacterial strain BL-gB+.
Cut pC-SaS and pG-gB with
Nco
I and
Spe
I. Recovery
and purify 11 kb fragment from pC-SaS and 3.2 kb
fragment from pG-gB with a recovery kit (Promega).
Ligate fragments with T4 DNA ligase (Promega) and
transform ligated plasmid into the competent cells of
the
E. coli
BL5α. After proving
glg
B being ligated
downstream of 35S promoter in pC-SaS, transform the
ligated plasmid into competent cells of the agrobacterial
to obtain recombined
Agrobacterial
LBA-gB containing
recombined pC-gB.
3.4 Transformation of
glg
B into potato
Cut 1 cm stem segments with axillary bud from
genotype DXY and ZHB as the explants, and
pre-culture on MS media for 2 days. According to the
protocol (Anne et al., 1994), after plantlet in glass
tuber grows up to 8~10 cm at height, transfer it into