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61
the peat soil and grow for 30 days under light density
of 20 000 Lx, 25
℃
and with 12 h irradiation per day.
Then move and grow plantlets under the same
condition but with 8h irradiation per day. Minitubers
were harvested after 30 days.
3.5 Screening for
glg
B transgenic lines
Isolate genome DNA using CTAB method with leaves
from two groups of transformants. With
glg
B primer
cloned from the JM109 genome DNA, PCR was done
as described above. PCR product was tested with 1%
agarose gel electrophoresis.
glg
B probe was made as instruction DIG labeled kit
(Mylab, Ltd. Beijing). Southern blot was carried out
following Molecular Cloning: A Laboratory Manual
(
Third Edition
) (CSH Press 2001). Hybridization
signal on HyBond N+Membrane was tested with Dig
Hybridization assay kit (Mylab, Ltd. Beijing). Image
of signal on the membrane was taken with digital
cameral.
3.6 RT-PCR test of
glg
B expression in transgenic
lines
Leaf DNA was extracted as described above from
each of the transgenic lines. With
glg
B prime as above,
RT-PCR was done as the instruction of RT-PCR kit
(Takara).
3.7 Staining starch from the transgenic lines
Extract starch with method as described by Kuipers et
al (1994), 0.02% starch suspension at 20
℃
was stained
with 5% Lugol.
3.8 Assay of amylose contents
Assay of amylose contents was done with the method
as described by Kuipers et al (1994).
3.9 Assay of starch viscosity
To measure the dynamic viscosity of tuber starch, the
following reactions were made: add 3 g of purified
starch (water contents 16%) in ddH
2
O to make 3%
water suspension
（
w/v
）
.Heat up suspension to 70
℃
and incubate at 70
℃
for 15 min for gelatinization.
Add 10 mL the gelatinized suspension to a capillary
viscometer in a device kept constant at 60
℃
. The time
required for the suspension to fully pass a 3 mm
interstice in the viscometer was recorded as t
1
.
Dynamic viscosity was calculated with the formula
η=η
r
×t
1
/t
0.
η
r
is the solvent viscosity, and here η
r
=
0.469 mPa.s when ddH
2
O goes through the 3 mm
interstice at 60
℃
, t
0
is the time required for equal
volume solvent (ddH
2
O) to flow through the interstice.
All data was analyzed by a statistic tool, DPS ver. 9.50
(Data Processing System, Ruifeng, Hangzhou, China).
Authors' Contributions
YXL designed the experiment andprimers used, performed data
analysis, wrote and revised the manuscript and supervised
performance of the project. LJF performed
glg
B isolation,
vector construction and
glg
B function confirmation in
E. coli
.
FXJ performed
glg
B transformation into potatoes, screening for
transgenic lines expressing
glg
B and phenotype assay. CXQ,
CQ and HQ involved partially in the experiments above.
Acknowledgements
This work was jointly supported by National Sci-Tech Support
Program in China (2009BADC5B01, to Yao XL) and National
Natural Science Foundation of China (NSFC 30660079,
31160236, To Yao XL).
References
Anne J.K., Angela M.S., Vermeesch B.J., de Vries, Evert J., and Richard
G.F.V., 1996, Expression of
Escherichia coil
branching enzyme in
tubers of amylose-free transgenic potato leads to an increased
branching degree of the amylopectin,
The Plant Journal, 10(1): 83-90
http://dx.doi.org/10.1046/j.1365-313X.1996.10010083.x
PMid:8758980
Haugen T.H., Ishaque A., and Preiss J., 1976, Biosynthesis of bacterial
glycogen, characterization of the subunit structure of
Escherichia coli
B glucose-1-phosphate adenylyltransferase (EC 2.7.7.27), The Journal
of Biological Chemistry, 251(24): 7880-7885 PMid:826540
Jobling S.A., Westcott R.J., Tayal A., Jeffcoat R., and Schwall G.P., 2002,
Production of a freeze-thaw-stable potato starch by antisense
inhibition of three starch synthase genes, Nat Biotechnol, 20(3):
295-299 http://dx.doi.org/10.1038/nbt0302-295 PMid:11875432
Kozlov S.S., Blennow A., Krivandin A.V., and Yuryev V.P., 2007, Structural
and thermodynamic properties of starches extracted from GBSS and
GWD suppressed potato lines, International Journal of Biological
Macromolecules, 40(5): 449-460 http://dx.doi.org/10.1016/j.ijbiomac.
2006.11.001 PMid:17188347
Kuipers G.J., Jacobsen E., and Visser R.G.E., 1994, Formation and
deposition of amylose in the potato tuber starch granule are effected
by the reduction of granule-bound starch synthase expression, The
Plant Cell, 6(1): 43-52 http://dx.doi.org/10.1105/tpc.6.1.43 PMid:12244219
http://dx.doi.org/10.2307/3869673 PMid:12244219 PMCid:160414
Tiessen A., Hendriks J.H.M., Stitt M., Branscheid A., Gibon Y., Farré E.M.,
and Geigenberger P., 2002, Starch synthesis in potato tubers is