To construct plasmids for the wild-type α′, DNA consisting of the α′ promoter (−963 to −1) and the α′-coding region was inserted between BamHI and KpnI sites of pBI101 (Clontech, Palo Alto, CA, USA), whose SacI site had been replaced by a KpnI site by means of inserting a KpnI linker (TaKaRa, Tokyo, Japan). The promoter region and the signal peptide and propeptide coding region of α′ were amplified from genomic DNA of soybean (Glycine max L. cv. Wasesuzunari), which had been prepared from young leaves by the cetyltrimethylammonium bromide (CTAB) nucleic acid extraction procedure (Rogers and Bendich, 1988), by PCR using the following primers: primer 1, 5′-GGGGGATCCGTTTTCAAATTTGAATTTTAATGTGTGTTGTAA-3′ (underline indicates a BamHI site introduced upstream of −963 of α′ promoter); primer 2, 5′- CTTAAGGAGGTTGCAACGAGCGTGG-3′. The extension and core coding region of α′ was amplified using α′ cDNA (Maruyama et al., 1998) as a template and the following primers: primer 3, 5′-GTGGAGGAAGAAGAAGAATGCGAG-3′; and primer 4, 5′-CGCGGTACCCGATACTTTCCTCGCTCACT-3′ (underline indicates a KpnI site introduced at 64 bases downstream of stop codon). Both amplified fragments were inserted together between BamHI and KpnI sites of pBluescript SK (Stratagene, La Lolla, CA, USA), creating pBSα′, and then the BamHI–KpnI fragment of pBSα′ was inserted into pBI101 with BamHI/KpnI sites to construct pBIα′.
To construct plasmids for α′ derivatives, ΔPropep, ΔExt and Core, we used the following primers: primers 1 and 2 for the promoter region and the signal and propeptide coding region using genomic DNA as a template for ΔExt; primers 1 and 5 (5′-AATGCCAAATGAGACAGAAACTGATGC-3′) for the promoter region and the signal peptide coding region using genomic DNA as a template for core and ΔPropep; primers 3 and 4 for the extension and core coding region using α′ cDNA as a template for ΔPropep; and primers 4 and 6 (5′-CGAAGACATAAGAATAAGAACCCTTTTC-3′) for the core coding region using α′ cDNA as a template for Core and ΔExt. Two of the PCR products were inserted together between BamHI and KpnI sites of pBluescript to construct pBSΔPropep, pBSΔExt, and pBSCore, and then the BamHI–KpnI fragments were inserted into pBI101 with BamHI/KpnI sites to construct pBIΔPropep, pBIΔExt, and pBICore.
According to the following procedures, plasmids for ΔCT6 and ΔCT10 were constructed. By PCR, a DNA fragment from a SacI site (+1396 from the initiation codon) in the core coding region to 18 (ΔCT6) or 30 (ΔCT10) base pairs upstream of the stop codon were amplified using the following primers: primer 7 (5′-GGACTTGGATGTCTTCCTCAGTG-3′) and primer 8 (5′-TGAAGACAAAGGACCCTTTCTTC-3′) for ΔCT6; and primers 7 and 9 (5′-ACCCTTTCTTCCCTTGTTCCC-3′) for ΔCT10 using pBSα′ as a template. Primer 10 (5′-TGAATAAGTATGTAGTACTAAAATTATG-3′; underline indicates a stop codon) and primer 11 (5′-CGCTTTCTTCCCTTCCTTTCTCGC-3′) were used for amplifying from the stop codon to a DraIII site which is on the f1(–) origin of pBSα′. These PCR fragments were inserted between SacI and DraIII sites of pBluescript SK, and then the SacI–KpnI fragments were inserted into pBIα′ with SacI/KpnI sites to construct pBIΔCT6 and pBIΔCT10.
pBSΔGlyc was constructed by means of mutation of the sites corresponding to two N-linked glycosylation signals of pBSα′. Codons for Asn 277 and Asn 533 were replaced with codons for Ser and Thr, respectively. Before mutation, we confirmed that these replacements do not disturb the folding of this derivative based on the three-dimensional structure of β homotrimers of β-conglycinin (Maruyama et al., 2001). We used pBSα′ as a template and the following primers: primer 12 (5′-GAACCAAGCATGCCACGCTCGTTGC-3′) and primer 13 (5′-AGTCCCGCTAAGGATAACGATGAGG-3′; underline indicates the codon for Ser substituted for Asn) were used for mutation of first glycosylation site, and primer 14 (5′-GCCATTCTTACCTTGGTGAACAACG-3′) and primer 15 (5′-GAAATTCAGATCTGAGGTAGCGGTG-3′; underline indicates the codon for Thr substituted for Asn) were used for mutation of second glycosylation site. Both amplified fragments were inserted together between AflII in the propeptide coding region and BglII sites in the core coding region of pBSα′ to construct pBSΔGlyc, and then the BamHI–KpnI fragment was inserted into pBI101 with BamHI/KpnI sites to construct pBIΔGlyc.
To construct the plasmid for CoreΔGlyc, we used pBSΔGlyc as a template and the following primers: primers 5 and 16 (5′-CAGTTACTTATCCTTCCTCCA-3′) for amplifying a fragment containing the region from an EcoT22I site in the promoter region to the signal peptide coding region, and primers 4 and 6 for amplifying the core coding region. These PCR fragments were inserted together between EcoT22I and KpnI sites of pBSα′ to construct pBSCoreΔGlyc, and then the BamHI–-KpnI fragment was inserted into pBI101 with BamHI/KpnI sites to construct pBICoreΔGlyc.
According to the following procedures, we constructed a plasmid for spGFP. By PCR, we prepared fragments of the α′ promoter and the signal peptide coding region using primers 1 and 5, and pBSα′ as a template. We also prepared a fragment of a GFP coding region using primer 17: 5′-GTGAGCAAGGGCGAGGAGCTGTTCA-3′ and T3 promoter primer, and GFP (S65T)/pBluescript SK (Chiu et al., 1996) as a template, kindly gifted by Dr Y. Niwa, University of Shizuoka. These fragments were inserted together between BamHI and KpnI sites of pBluescript to construct pBSspGFP, and the BamHI–KpnI fragment was inserted into pBI101 with BamHI/KpnI sites to construct pBIspGFP.
According to the following procedures, we constructed a plasmid for GFP-CT 24. We used primer 18 (5′-GCTCTAGAGCGTGAGCAAGGGCGAGGAGCTGTTCA-3′; underline indicates an XbaI site introduced upstream of the codon for the second residue Val of GFP), primer 19 (5′-CTTGTACAGCTCGTCCATGCCGT-3′) and GFP (S65T)/pBluescript SK as a template for amplifying a fragment coding GFP, and primers 11 and 20 (5′-CAGCCTCAGCAGAAAGAGGAGGGGAA-3′), and pBSα′ as a template for amplifying C terminal 24 amino acids coding region of α′ cDNA. Both fragments were inserted together between XbaI and KpnI sites of pBluescript to construct pBSgfp-ct24. Then, using primer 17 and T3 promoter primer, a fragment of GFP having the C-terminal region of α′ was amplified from pBSgfp-ct24. The fragment was inserted with the fragment of the α′ promoter and the signal peptide coding region between BamHI and KpnI sites of pBluescript to construct pBSGFP-CT24. Then, the BamHI–KpnI fragment was inserted into pBI101 with BamHI/KpnI sites to construct pBIGFP-CT24.
According to the following procedures, we constructed a plasmid for spmGFP containing four contiguous Glys at its C-terminus. We used primer 21 (5′-TAAAGCGGCCGCCCGGCTGCAG-3′; italics indicates the stop codon), primer 22 (5′-TCCTCCTCCTCCCTTGTACAGCTCGTCCAT-3′, underline indicates codons for glycines introduced before the stop codon) and pBSspGFP as a template. The amplified fragment was self-ligated to construct pBSspmGFP.
According to the following procedures, we constructed plasmids for mGFP-CT24 and mGFP-CT10 having the C-terminal 24 and 10 amino acids of α′, respectively. We used primer 22 and each of primer 20 or primer 23 (5′-CCTTTGTCTTCAATTTTGAGGGCTTTTTACTGA-3′), and pBSGFP-CT24 as a template. The amplified fragments were self-ligated. To exchange the 3′ untranslated region of these plasmids, which were derived from α′, with the nopaline synthase (nos) terminator, a DNA fragment from a BstYI site (+572 from the initiation codon) in the GFP region of these plasmids to the stop codon was amplified using the following primers: primer 24 (5′-GGTGAACTTCAAGATCCGCCAC-3′, underline indicates a BstYI site), primer 25 (NotI) (5′-ATAGTTTAGCGGCCGCTCAGTAAAAAGCCCT-3′; underline indicates introduced NotI site, and italics indicates the stop codon). The amplified fragments were inserted into pBSspGFP with BstYI/NotI sites to construct pBSmGFP-CT24 and pBSmGFP-CT10.
To construct a plasmid for mGFP-CT24Δ10, we used primer 24 and primer 26 (5′-ATAGTTTAGCGGCCGCTTAACCCTTTCTTCCCTTGTT-3′; underline indicates NotI site, and italics indicates the stop codon introduced after the codon for the eleventh amino acids from the last), and pBSmGFP-CT24 as a template. The amplified fragment was inserted into pBSspGFP with BstYI/NotI sites to construct pBSmGFP-C24Δ10.
Sequences of the DNA regions inserted into pBluescript were confirmed by ABI Prism 310 DNA Analyzer (Applied Biosystems, Foster City, CA, USA) before transfer to pBI101. All pBI plasmids were transformed into A. tumefaciens LBA4404 strain by electroporation using Gene Pulser Transfection Apparatus (Bio-Rad, Hercules, CA, USA).
Total protein (20 µg) of each crude cell extract was dissolved in SDS sample buffer composed of 50 mm Tris–HCl (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol, 1% (v/v) 2-mercaptoethanol, and 0.004% (w/v) Bromophenol Blue. After boiling, the samples were subjected to SDS–PAGE on 11% polyacrylamide gel. The separated proteins on gels were transferred electrophoretically to a nitrocellulose membrane (0.45 µm; Schleicher & Schuell Inc., Dassel, Germany) and detected with anti-α′ subunit serum or anti-GFP serum (Invitrogen, Carlsbad, CA, USA) followed by a goat antirabbit IgG-alkaline phosphatase conjugate (Promega, Madison, WI, USA).
Arabidopsis T2 dry seeds were imbibed in water for 20 min before processing. Seeds were vacuum-infiltrated for 1 h with a fixative containing 50 mm sodium phosphate buffer (pH 7.2) and 1.5% (w/v) glutaraldehyde. The seeds were cut into halves and fixed for another 2 h in the fixative, followed by dehydration in an ethanol series, and embedded in epoxy resin (Quetol-812; Nisshin EM, Tokyo, Japan). Ultrathin sections were mounted on copper grids previously coated with carbon-coated formvar films, etched with 3% (v/v) aqueous hydrogen peroxide for 10 min, and immersed in blocking solution composed of PBS containing 1% (w/v) BSA for detection of α′ or 5% (w/v) BSA for detection of 11S globulin for 30 min at room temperature. Then, they were incubated with antiserum against α′ (1 : 2000) diluted in blocking solution or antiserum against glycinin (1 : 50) diluted with diluting solution composed of PBS containing 1% (w/v) BSA and 0.25% (v/v) Tween 20 for 1 h at room temperature. After washing with blocking solution or diluting solution, sections were incubated for 30 min at room temperature with antirabbit IgG secondary antibody conjugated to 15 nm colloidal gold (Amersham Biosciences, Piscataway, NJ, USA) diluted 1 : 25 in blocking solution or diluting solution. The sections were rinsed with PBS and distilled water, and then stained with 4% (w/v) uranyl acetate and lead citrate. After staining, the sections were examined with a transmission electron microscope (model H-700H; Hitachi, Tokyo, Japan) at 100 kV. Control experiment was performed by omitting the primary antibody.