Iranchild1: Створена сторінка: Morphology observations were carried under a stereomicroscope (SZX16, Olympus, Tokyo). Samples were fixed in 2.5% glutaraldehyde solution. Fixed samples were de...

2017-05-01T16:49:37Z

Створена сторінка: Morphology observations were carried under a stereomicroscope (SZX16, Olympus, Tokyo). Samples were fixed in 2.5% glutaraldehyde solution. Fixed samples were de...

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Morphology observations were carried under a stereomicroscope (SZX16, Olympus, Tokyo). Samples were fixed in 2.5% glutaraldehyde solution. Fixed samples were dehydrated with gradual ethanol series, dried by critical-point drying method using liquid carbon dioxide (Model HCP-2, Hitachi, Tokyo), gold-coated with an Edwards E-1010 ion sputter coater (Hitachi, Tokyo), and then observed using a S-3000N variable pressure scanning electron microscope (Hitachi, Tokyo). The dp1 locus was mapped by using an F2 population of dp1�C2 and Sheng47 (spp. indica). The locus was mapped [http://en.wikipedia.org/wiki/Diglyceride diglyceride] to a region between cleaved-amplified polymorphic sequence (CAPS) markers M4 and M6 on the short arm of chromosome 6 ( Luo et al., 2005). We further developed two new CAPS markers M9 and dM1 in this region (Table S2) to narrow the locus to a 10?kb region between M4 and dM1 ( Fig.?6A). The sdp1 locus was mapped by using an F2 population of sdp1 and Minghui 63 (spp. indica). The locus was mapped to a 92?kb region between two sequence-tagged site markers, S14533 and S14625 (Fig. S6). Primer sequences used for vector construction [http://www.selleckchem.com/products/a-1210477.html A-1210477 in vitro] are listed in Table S2 in the Supplementary data. For complementation of the dp1�C2 mutant, a 9292?bp genomic fragment containing the entire DP1 coding sequence, 5728?bp of the 5�� upstream region and 2577?bp of the 3�� downstream region was digested form PAC clone P0548D03, and cloned into the pCAMBIA1300 vector to generate plasmid p1300-DP1. For RNA interference analysis, a fragment of the DP1 cDNA (864?bp�C1276?bp of the coding region) was amplified and cloned into pUCCRNAi vector by forward and reverse insertions. The entire fragment was subcloned into pCAMBIA1300A under [http://www.selleckchem.com/products/ldn193189.html LDN193189] the rice ACTIN1 promoter. For pDP1::GUS and pDP1::DP1-GFP (green fluorescent protein), specific primers with suitable adaptors were designed to amplify relative sequences (Table S2), and cloned into pCAMBIA1301 or CAMV35S-sGFP(S65T)-Nos ( Niwa et al., 1999), respectively. These vectors were subsequently transformed into calli derived from mature rice seeds through Agrobacterium-mediated methods ( Hiei et al., 1994). Total RNA was extracted using TRIZOL (Invitrogen, Carlsbad). The RNA was pre-treated with RNase-free DNase I (Takara, Shiga), and first-strand cDNA was synthesized from 2?��g of total RNA using reverse transcriptase (M-MLV, Promega, Madison). The reverse transcription product was used for PCR with gene specific primers (Table S2). Rice ACTIN1 was used as the internal reference. For qRT-PCR, SYBR Green I was added to the reaction system and run on a Chromo 4 real-time PCR detection system (Bio-Rad, Hercules) according to the manufacturer's instructions. Three replicates were carried out for each gene, and each analysis was biologically repeated at least twice. Student's t-test was used to determine significant changes (P?

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Iranchild1: Створена сторінка: Morphology observations were carried under a stereomicroscope (SZX16, Olympus, Tokyo). Samples were fixed in 2.5% glutaraldehyde solution. Fixed samples were de...