Page 15 - Molecular Plant Breeding

Molecular Plant Breeding 2011, Vol.2, No.8, 48 -59
http://mpb.sophiapublisher.com
58
reading frame of bar gene and nos terminator from
pCB1 digested with EcoRⅤ was used as probe for
bar gene. The linear fragment (1.12 kb) from pCB1
after digestion with Hind Ⅲ and Pst Ⅰ , which
comprises cecropin B coding region and Pin
terminator was used as probe for cecropin B gene. The
probe structure and sites on pCB1 are illustrated in
Figure 3.
The probe labeled by random priming was conducted
using DIG DNA Labeling Kit (Roche Company)
according to the manufacture’s instructions. Southern
blot hybridization and detection was carried out using
DIG Luminescent Detection Kit (Roche Company)
according to the manufacture’s instructions.
3.4 Northern blot hybridization
Leaf tissue (0.5~1g) was ground in liquid nitrogen.
The dispersed tissue was used for total RNA
extraction using TRIzol Kit (Invitrogen Company)
according to the manufacture’s instructions. Extracted
RNA was initially checked for quality and quantity on
normal 1% agarose gel. After electrophoresis on 1.2%
formaldehyde-agrose gel, the gel was washed for
10 min in sterile water to remove the formaldehyde.
The RNA was denatured in 0.05 mol/L NaOH and
blotted onto Amersham N+ Hybond membranes in
20×SSC according to the Southern blotting method
(Meyer et al., 1995). The DNA probe of cecropin B
gene described as above was used for Northern
hybridization.
Northern blot hybridization and detection was carried
out using DIG Luminescent Detection Kit (Roche
Company) according to the manufacture’s instructions.
Authors’ contributions
Yan Zhao conducted the major part of this study including experimental
design, Southern and Northern hybridization experiment, and manuscript
preparation. Longbiao Guo and Huizhong Wang conducted the rice material
hybrid design and the crossing experiment. Danian Huang participated in
experimental design.
Acknowledgements
This work was supported by grants from the National Nature Science
Foundation of China (No: 30871511&30771317), the key program from the
Ministry of Agriculture of China for Creation of New Transgenic Organism
Varieties (Nos: 2008ZX0810-003 &2009ZX08001-022B), the post-doctoral
foundation of China (No.20090450477).
References
Altpeter F., Baisakh N., Beachy R., Bock R., Capell T., Christou P., Daniell H.,
Datta K., Datta S.J., Dix P., Fauquet C., Huang N., Kohli A., Mooibroek H.,
Nicholson L., Nguyen T.T., Nugent G., Raemakers K., Romano A.,
Somers D., Stoger E., Taylor N., and Visser R., 2005, Particle
bombardment and the genetic enhancement of crops: myths and realities,
Mol. Breed., 15(3): 305-327 doi:10.1007/s11032-004-8001-y
Chen H., Tang W., Xu C.G., Li X.H., and Zhang Q., 2005, Transgenic indica
rice plants harbouring a synthetic cry2A* gene of Bacillus thuringiensis
exhibit enhanced resistance against rice lepidopteran pests, Thero. Appl.
Genet., 111(7): 1330-1337 doi:10.1007/s00122-005-0062-8 PMid:16187120
Fojtová M., Bleys A., Bedřichová J., Houdt H.V., Křížová K., Depicker A., and
Kovařík A., 2006, The transsilencing capacity of invertedly repeated
transgenes dependends on their epigenetic state in tobacco, Nucleic Acids
Res., 34(8): 2280-2293 doi:10.1093/nar/gkl180 PMid:16670434 PMCid:
1456325
Horvath H., Jensen L.G., Wong Q.T., Kohl E., Ullrich S.E., Cochran J.,
Kannangara C.G., and Von Wettstein D., 2001, Stability of transgene
expression, field performance and recombination breeding of transformed
barley lines, Thero Appl Genet, 102(1): 1-11 doi:10.1007/s001220051612
Hua Z.H., Zhu X.F., Wu M.G., Yu Y.C., Zhao Y., Gao Z.Y., Yan M.X., and
Qian Q., 2003, Inheritance of exo-genes integrated into the rice genomes,
Acta Agronomica Sinica, 29(1): 44-48
Huang D.N, Li J.Y., Zhang S.Q., Xue R., Yang W., Hua Z.H., Xie X.B., and
Wang X.L., 1998, New technology to examine and improve the purity of
hybrid rice with herbicide resistant gene, Chin. Sci. Bull., 43(9): 784-787
doi:10.1007/BF02898961
Huang D.N., Zhu B., Yang W., Xue R., Xiao H., Tian W.Z., Li L.C., and Dai
S.H., 1996, Introduction of cecropin B gene into rice (Oryza sativa L) by
particle gun bombardment and analysis of transgenic plants, Science in
China (Series C), 39(6): 652-661
Iyer L.M., Kumpatla S.P., Chandrasekharan M.B., and Hall T.C., 2000,
Transgene silence in monocots, Plant Mol. Biol., 43(2-3): 323-346
doi:10.1023/A:1006412318311 PMid:10999414
Khush G.S., 2005, What it will take to feed 5.0 billion rice consumers in 2030,
Plant Mol. Biol., 59(1): 1-6 doi:10.1007/s11103-005-2159-5 PMid: 16217597
Kohli A., Griffiths S., and Palacios N., 1999, Molecular characterization of
transforming plasmid rearrangements in transgenic rice reveals a
recombination hotspot in the CaMV35S promoter and confirms the
predominance of microhomology mediated recombination, Plant J., 17(6):
591-601 doi:10.1046/j.1365-313X.1999.00399.x PMid:10230059
Kohli A., Twyman R.M., Abranches A., Wegel E., Stager E., Christou P., and
Stoger E., 2003, Transgene integration, organization and interaction in
plants, Plant Mol. Biol., 52(2): 247-258 doi:10.1023/A:1023941407376
PMid:12856933
Kumpatla S.P., Teng W., Buchholz W.G., and Hall T.C., 1997, Epigenetic
transcriptional silencing and 5-azacytidine-mediated reaction of a complex
transgene in rice, Plant Physiol., 115: 361-373 doi:10.1104/pp.115.2.361
PMid:9342860 PMCid:158494
Lindsay H., Adams R.L., 1996, Spreading of methylation along DNA, Biochem.
J., 320(Pt2): 473-478 PMid:8973555 PMCid:1217954
Lu Y.J., and Zheng K.L., 1992, A simple method for isolation of rice DNA,
Chinese J. Rice Sci., 1: 47-48 (in Chinese with an English abstract)
Matzke A.J.M., and Matzke M.A., 1998, Position effect and epigenetic silencing
of plant transgenes, Curr. Opin. Plant Biol., 1(2): 142-148 doi:10.1016/
S1369-5266(98)80016-2
Meyer P., 1995, Understanding and controlling transgene expression, Trends
Biotechnol., 13(9): 332-337 doi:10.1016/S0167-7799(00)88977-5
Peng S., Laza R.C., Visperas R.M., Sanico A.L., Gassman K.G., and Khush
G.S., 2000, Grain yield of rice cultivars and lines developed in Philippines
since 1996, Crop Sci., 40(2): 307-314 doi:10.2135/cropsci2000.402307x
Popelka J.C., and Altpeter P., 2003, Evaluation of rye (Secale cereale L.) inbred
lines and their crosses f or tissue culture response and stable genetic
transformation of homozygous rye inbred line L22 by biolistic gene
transfer, Thero. Appl. Genet., 107(4): 583-590 doi:10.1007/s00122-