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Supplemental materials and methods
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Construction of the SBEI RNAi vector p13dsSBE1
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The full-length SBEI cDNA (Figure S1a) was first cloned by RT-PCR from total RNA
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isolated from the developing rice seeds, and the Xba I site was added at both the ends of
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cloned cDNA (Chen et al., Rice Science, 2004, 11(3): 81–85). The 680-bp Xba I-Bam HI
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fragment at the 5’-end of cloned SBEI cDNA (Figure S1a) was used for construction of the
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SBEI RNAi vector. First, the 1.3-kb rice glutelin GluA-3 (Gt1) promoter, the 103-bp of
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intron 2 of rice Gt1 gene and the NOS terminator were inserted into the multiple cloning
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sites of the T-DNA region of the binary vector pCAMBIA1300 (kindly provided by Prof.
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Jefferson, CAMBIA, Australia), which resulted in the intermediate vector p1011 (Liu et al.,
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unpublished data). At both ends of Gt1 intron 2 in p1011, a multiple cloning site was added
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for further cloning of the sense or antisense target fragment. The 680-bp SBEI cDNA
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fragment was then inserted between the rice Gt1 promoter and intron 2 at the antisense
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orientation, while at the sense orientation between the intron 2 and NOS terminator. The
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rice Gt1 promoter would be required for high level of expression of the targeted dsRNA,
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specifically in rice endosperm. The resultant binary vector was namedp13dsSBE1 (Figure
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S1c).
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Construction of the SBEIIb RNAi vector p13dsSBE3
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The full-length SBEIIb cDNA (Figure S1b) was cloned as described above, and the Xba I
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site was also added at both the ends of cloned cDNA (Chen et al., Rice Science, 2004, 11(3):
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81–85). For RNAi construction, the 250-bp Bgl II-Xba I fragment at the 3’-end of cloned
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SBEIIb cDNA (Figure S1b) was then inserted between the rice Gt1 promoter and intron 2 at
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the antisense orientation in vector p1011 as shown above, while at the sense orientation
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between the intron 2 and NOS terminator. The resultant plasmid was named p13dsSBE3
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(Figure S1c).
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Construction of the vector p13aSBE13 containing both antisense SBEI and SBEIIb
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fragments
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For this double antisense SBE construct, the 680-bp Xba I-Bam HI fragment at the 5’-end
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of cloned SBEI cDNA (Figure S1a) and the 997-bp Sac I-Kpn I fragment at the 5’-end of
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cloned SBEIIb cDNA (Figure S1b) were used. Both fragments, at an antisense orientation,
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were inserted into p1011 between the rice Gt1 promoter and intron 2 and between the
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intron 2 and NOS terminator, respectively. The resultant construct was named p13aSBE13
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(Figure S1c).
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PCR and Southern blot analyses of transgenic rice plants
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Total genomic DNA was extracted from rice leaves as described by Murray and Thompson
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(Murray MG and Thompson WF. Rapid isolation of high molecular weight plant DNA. Nucl
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Acids Res 1988, 8: 4321-4325). Primers INT-F (5’-CCTCGTAATCAATTGTTAGGAT-3’)
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and NOS-R (5’-GACCGGCAACAGGATTCAAT-3’) were used for identification of
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transgenic rice, which were located in the Gt1 intron 2 and NOS terminator of the
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constructed vectors, respectively, as shown in Figure S1c. The sizes of PCR products from
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transgenic rice plants with constructs p13dsSBE1, p13dsSBE3, and p13aSBE13 (Figure S1c)
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were 763 bp, 333 bp, and 1080 bp, respectively (Figure 1a, d).
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For Southern blots, ten g aliquots of total DNA were digested with restriction
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endonuclease Bam HI (TaKaRa) and were separated on an agarose gel and transferred to
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Hybond-N+ nylon membranes (Roche). A fragment of SBEI cDNA was labeled with
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digoxigenin (DIG) by using a DIG nucleic acid labeling kit (Roche), then as a hybridization
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probe. Hybridization and signal detection were carried out according to the manual of the
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Roche Company.
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