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1 Okagaki R J, Wessler S R. Comparison of non-mutant and mutant
waxy genes in rice and maize. Genetics, 1988, 120: 1137�1143
2 Wang Z, Wu Z, Xing Y, et al. Nucleotide sequence of rice waxy
gene. Nucl Acids Res, 1990, 18: 5898
3 ���, ���, �
, �. �
waxy ���������
��������� . !: 21 "#��$%&'(). *+:
,-./012, 1999. 206�213
4 Terada R, Nakajima M, Isshiki M, et al. Antisense waxy genes
with highly active promoters effectively suppress Waxy gene
expression in transgenic rice. Plant Cell Physiol, 2000, 41: 881�
888
5 345, ���, ���, �. 67.89:�;<=�>?�
@�� . AB./� , 2001, 22(1): 1�6
6 Murray M G, Thompson W F. Rapid isolation of high molecular
weight plant DNA. Nucl Acid Res, 1980, 8: 4321�4325
7 Sambrook J, Russell D. Molecular Cloning: A Laboratory Manual.
3rd ed. New York: Cold Spring Harbor Laboratory Press, 2001,
6.33�6.62.
8 Jefferson R. A. Assaying chimeric genes in plants: The GUS
fusion system. Plant Mol Biol Rep, 1987, 5: 387�405
9 Juliano B O. A simplified assay for milled rice amylose. Cereal
Sci Today, 1971, 16: 334�360
10 Murashige T, Skoog F. A revised medium for rapid growth and
bioassays with tobacco tissue culture. Physiol Plant, 1962, 15:
473�479
11 Hiei Y, Ohta S, Komari T, et al. Efficient transformation of rice
(Oriza sativa L) mediated by Agrobacterium and sequence
analysis of the boundaries of the T-DNA. Plant J, 1994, 6: 271�
282
12 CD, �
, EFG, �. �H�������$%IJKL
M�� . !: NOP, �
, QRS, �T. UVWXYZ
[\$%� ](. ^+: AB_F`a012, 1990. 68�74
13 vander Krol A R, Stuitje A R. Antisense genes in plants: an
overview. Gene, 1988, 72: 45�50
14 Grierson D, Fray R G, Hamiltton A T, et al. Does co-suppression
of sense genes in transgenic plants involve antisense RNA?
Trends Biotechnol, 1991, 9: 122�123
15 Shimada H, Tada Y, Kawasaki T, et al. Antisense regulation of the
rice waxy gene expression using a PCR-amplified fragment of the
rice genome reduce the amylose content in grain starch. Theor
Appl Genet, 1993, 86: 665�672
16 Juliano B O. Criteria and test for rice grain quality. In: Juliano B
O, ed. Rice chemistry and technology. St Paul Minn: American
Association of Cereal Chemists Inc, 1985. 443�513
17 ���, �bc, �
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Wx ��d��e[$%
If�Hgh�LMij . ,-./_F, 2002, 35(2): 117�
122
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