BN-PAGE实验操作步骤.doc

Supplemental MaterialAppendix S1. Experimental proceduresPigment analysisPrior to pigment extraction, each sample was weighed. Seedlings were frozen in liquid nitrogen and ground to powder with a high-speed mixer mill (model MM301, Retsch, ). Total chlorophyll and carotenoid were extracted from 30 seedlings with 100% acetone. The debris was removed by centrifugation at 10,000 rpm for 10 min. Absorbances of the supernatant were measured with a Beckman DU650 spectrophotometer (), and the contents of the pigments were determined according to Lichtenthaler and Wellburn (1983).Anthocyanin extraction and quantification was performed as described previously (Kim et al., 2003). In brief, anthocyanin was extracted from 40 seedlings with 300 L of acidified (1% HCl) methanol overnight at 4C in dark conditions. The extract was separated by the addition of 200 L of water and 200 L of chloroform, followed by centrifugation for 5 min at 3,000 rpm. The absorbance of the upper phase was determined spectrophotometrically at 530 nm and 657 nm, and the anthocyanin content was calculated as A530 0.33A657. For Pchlide analysis, wild-type and heterozygous atterC-1 seeds were sown onto MS medium and grown in the dark for 4 d at 22C. At the end of the dark treatment, plates were exposed to light for 20 h to select the pigment-deficient homozygous atterC-1 seedlings. The selected mutant seedlings were then transferred to fresh MS medium on a clean bench. The wild-type and mutant seedlings were kept in darkness again for an additional two days. Seedlings were harvested under a dim-green light, and Pchlide extraction was performed as described previously (Terry and Kendrick, 1999). Seedlings were homogenized as described above. Extracts were obtained from 80 seedlings. After extraction with 0.5 mL of cold acetone (0.1 M NH4OH (90/10, v/v), samples were centrifuged at 30,000 g for 10 min. The pellet was then re-extracted in 150 L solvent and centrifuged again. The supernatants were combined and washed twice with an equal volume and a one-third volume of hexane prior to spectrophotometric analysis. Fluorescence spectroscopy was performed using a Hitachi F-2000 (). Fluorescence was measured using an excitation/emission wavelength of 440/637 nm for Pchlide. All pigment measurements were repeated in three independent experiments.Protein extraction and immunoblot analysisProtein extraction was performed as described by Frick et al. (2003), except that the concentration of SDS was changed to 0.5%. Protein extracts were obtained from frozen seedlings ground in liquid nitrogen and thawed in homogenization buffer (0.0625 M Tris-HCl, pH 6.8; 0.5% w/v SDS; 10% v/v glycerol; 0.5% v/v -mercaptoethanol) at 95C for 10 min. After centrifugation at 16,000 g for 10 min, supernatant protein concentrations were determined with Bradford reagent (USB, ) using BSA as a standard, then separated by 10% SDS-PAGE. The proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, ) by electroblotting for 1 h at 90 V in 25 mM Tris, 0.2 M Glycine, and 20% (w/v) methanol. Blocking and immunodecorations were performed in Tris-buffered saline containing 5% nonfat dry milk. Protein gel blots were developed using a WEST-ZOL plus western blot detection system (iNtRON). Antisera used in the immunoblot assay were as follows: Anti-D1 (Cat. no. AS06124A, diluted 1:2000), anti-PasA (Cat. no. AS03025, diluted 1:5000), anti-Cyt f (Cat. no. AS06119, diluted 1:5000), anti-AtpB (Cat. no. AS05085, diluted 1:2000), anti-rbcL (Cat. no. AS03037, diluted 1:10000), anti-Tic40 (Cat. no. AS06149, diluted 1:5000) antibodies were purchased from AgriSera (). Antibodies used for the immunological detection of CAB (Cat. no. sc-12691, diluted 1:2000) and GFP (Cat. no. sc-9996, diluted 1:1000) were purchased from Santa Cruz Biotechnology (). The antiserum against Arabidopsis POR was kindly provided by G. Armstrong (Ohio State University, Columbus, OH). Anti-actin antibody (Cat. no. MA1-744, Affinity BioReagents, ) was used to confirm equal loading of proteins.Polysome analysisPolysomes were isolated as described previously by Barkan (1998). Frozen seedlings (0.2 g) were ground in liquid nitrogen, and 1 mL of polysome extraction buffer (0.2 M Tris-HCl, pH 9.0, 0.2 M KCl, 35 mM MgCl2, 25 mM EGTA, 0.2 M sucrose, 1% w/v Triton X-100, 2% w/v polyoxyethylene-10-tridecy ether, 100 mM -mercaptoethanol, 0.5 mg/mL heparin, 100 g/mL chloramphenicol, and 25 g/mL cycloheximide) was added. Sample was ground further until thawed and placed on ice for 10 min. Nuclei and insoluble materials were pelleted by centrifugation at 13,000 rpm for 5 min at 4C. Sodium deoxycholate was added to the supernatant to a final concentration of 0.5% (w/v), and the sample was placed on ice for 5 min. Remaining insoluble material was subsequently pelleted by centrifugation at 13,000 rpm for 15 min at 4C. The supernatant (0.5 mL) was layered onto 4-ml sucrose gradients and centrifuged with a Beckman SW 55 swing rotor at 45,000 rpm for 65 min at 4C. Four hundred and fifty microliters of each fraction was collected by gentle pipetting from the top of the gradient and added to 50 L of a solution consisting of 5% SDS and 0.2 M EDTA, pH 8.0. RNA was purified from each fraction by phenol:chloroform:isoamyl alcohol (25:24:1) extraction and subsequent ethanol precipitation. Finally, RNA samples were resuspended in 30 L of TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA), and 5 L of each fraction was examined by RNA gel blot analysis.Chloroplast isolation and subfractionIntact chloroplasts were isolated as previously described (Barneche et al., 2006) with little modification. Leaves were homogenized in ice-cold CIB buffer (0.45 M sorbitol, 50 mM Hepes-KOH pH 7.8, 2 mM EDTA, 0.1% BSA, 2.5 mM MgCl2, Protease Inhibitor Cocktail) using a mortar and pestle. The extract was filtered through two layers of Miracloth, debris were eliminated by centrifugation for 5 s at 1000 g, and the material was concentrated by centrifugation for 7 min at 1000 g. The pellet was resuspended in 1 mL of CIB and then layered on a discontinuous 40% (6 mL) / 80% (10 mL) Percoll gradient. After centrifugation for 15 min at 7000 g intact chloroplasts were isolated at the interface of the two layers. Chloroplasts isolated after two Percoll gradients were washed twice in HMS buffer (0.33 M Sorbitol, 50 mM Hepes-KOH pH 7.8, 2.5 mM MgCl2 and protease inhibitor cocktail) with centrifugation for 5 min at 1000 g. The chloroplasts were broken by osmotic lysis in 1 mL of lysis buffer (10 mM Hepes-KOH pH 7.8, 4 mM MgCl2, 1 mM PMSF, Protease Inhibitor Cocktail) with vortex, and incubated on ice for 10 minPreparation of chloroplast subfractions was carried out as previously described (Robinson and Barnett, 1988). After addition of 150 L of 80% (w/v) sucrose to the 1-mL lysed chloroplast, the mixture was layered on a discontinuous sucrose gradient of 0.98 M and 0.6 M sucrose, buffered in each case with 10 mM Hepes-KOH pH 7.8, 4 mM MgCl2, and then centrifuged with a Beckman SW 41 swing rotor at 90,000 g for 2 h. The stroma was obtained from the top fraction over 0.6 M sucrose. The envelope was collected at the interface between the layers of 0.6 M and 0.98 M sucrose and thylakoids as a pellet. The stroma fraction was precipitated with 10% trichloroacetic acid. In order to remove most of the soluble stromal proteins sequestrated in the envelope vesicles, envelope membrane preparations were further treated by sonication as previously described (Miras et al., 2002). The collected envelope fractions were diluted with 3 vols of lysis buffer containing 0.5 M NaCl and incubated for 30 min at 4C. Then, the mixture was sonicated for 10 s and stored for 15 min on ice before centrifugation with a Beckman SW 55 swing rotor at 72,000 g for 35 min at 4C, and envelope proteins recovered in the pellet. Oligonucleotide sequences and cDNA fragmentsMutant identification: P1 (5-CTTGGTGGATTAAG GAGACTTTTCA-3), P2 (5-ACCGCAGTGCCAACCTATTGA-3), LBa1 (5-TGGTTCACGTAGTGGGCCATCG-3), L1 (5-GTCTACATTCACGTCCAAATGGGGGC-3), and R1 (5-GTTCCAACCACG TCTTCAAAGCAAGTG-3). RNA gel blot and polysome analysis: NB-P1 (357-bp 5 coding region of AtTerC, NM_121251), 5-ATGAGCTTAGCTTCAGTTATCCACCACGGA-3, 5-AGTCTTGAAGGAAGTTTTGTAAGTTTCTTCTTG-3; NB-P2 (1155-bp full-length cDNA region of AtTerC ), 5-ATGAGCTTAGCTTCAGTTATCCACCACGGA-3, 5-TTAGCTGTCGCT GGATTTGTTTGTTAGGCTC-3; NB-P3 (329-bp 3 UTR region of AtTerC), 5-CTAAGAACTGAGAGCTGACTACTGAGAAAC-3, 5-CCAAAGGAATAATCTTATAAGATGAGC-3; psbA (X79898), 5-ATGACTGCAATTTTAGAGAGACGCGAAAG-3, 5-TTATCCATTTGTAGATGGAGCCTCAACAG-3; rbcL (U91966), 5-ATGTCACCACAAACAGAGACTAAAGCAAG-3, 5-CTACTCTTGGCCATCTAATTTATCGATGG-3; rbcS (NM_202369), 5-CACCGGTTAATTTCCCTTTGCTTTTGTG-3, 5-GTGAAACTAATTTTCTTAATGATTTCTTTAGCGAC-3; psaA (ATCG00350), 5-ATGATTATTCGTTCGCCGGAACCAG-3, 5-GCTGCTTTGTGATAATGGAACCAACCA-3; petA (ATCG00540), 5-ACCTTTTCTTGGATAAGGGAAGAGATTAC-3, 5-ACATCTTTATTAGTAGCAGGGTCTGGAGC-3; atpB (ATCG00480), 5-TCCTACTAC TTCAAATCCA GAGGTTTCGA-3, 5-CACCAAATACGGATACACCACCATGAGCT-3DFR (AJ251982), 5-TTGAAGGTGTTGATGAGAATCTAAAGAGCA-3, 5-ACCGTTACAATCACACGCGATACAAAATC-3. For CHS (M20308), a 1.3-kb HindIII fragment of pCHS3.9 (Feinbaum and Ausubel, 1988) was used. For CAB (AY045673), a 0.5-kb BamHI/BglII fragment of the RAFL 08-09-P03 clone obtained from RIKEN BRC was used. At18S ribosomal RNA (X16077) 5-GGTTGATCCTGCCAGTAGTCATATGC-3 and 5-GCCCTCCAATGGATCCTCGTTAAGG-3. Complementation analysis: (5-GCAGTCGACGATGAGGAACGAGAGGA-3 and 5-GCACTGCAGGAAAACATCAGATGTTCACCT-3). DNA gel blot analysis: 5-GAGACCTGATTTCATAGTTATCTCAATAGG-3 and 5-GTTACCGCAAATGCTATGTCACTGAG-3. For AtTerC coding region of 35S:AtTerC:GFP: 5-ACTAGTATGAGCTTAGCTTCAGTTATCCACCAC-3 and 5-ACTAGTGCTGTCGCTGGATTTGTTTGTTAGG-3Barkan, A. (1998) Approaches to investigating nuclear genes that function in chloroplast biogenesis in land plants. Methods Enzymol. 297, 38-57.Barneche, F., Winter, V., Crvecur, M. and Rochaix, J.D. (2006) ATAB2 is a novel factor in the signalling pathway of light-controlled synthesis of photosystem proteins. EMBO J. 25, 5907-5918. Feinbaum, R.L. and Ausubel, F.M. (1988) Transcriptional regulation of the Arabidopsis thaliana chalcone synthase gene. Mol. Cell. Biol. 8, 1985-1992.Frick, G., Su, Q., Apel, K. and Armstro