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mogana1991新虫 (初入文坛)
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论文的材料方法部分---英语翻译成汉语
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Plant Materials and Growth Conditions Arabidopsis thaliana (ecotype Columbia) was used as the wild type. Protoplasts were prepared from cultured Arabidopsis cells that had been subcultured and were incubated in medium containing Murashige and Skoog salts and 0.4 M mannitol, as described previously (Tamura et al., 2003). Tobacco (Nicotiana tabacum) BY-2 cells were cultured as described previously (Mitsuhashi et al., 2000). T-DNA insertion mutants (SALK_104728 [nup136-1] and SAIL_796_H02 [nup136-2]) were obtained from the ABRC at Ohio State University. Bioinformatics of the Arabidopsis Nucleoporins The data sets of the Arabidopsis nucleoporins were queried against The Arabidopsis Information Resource (TAIR) database (https://www. Arabidopsis.org), ATTED-II (https://atted.jp/), or GenomeNet (https://www. genome.jp/) to obtain annotations, functional assignments, structural information, and sequence relationships. Domain architectures were predicted using a domain analysis tool on the EMBL-EBI website (InterProScan; https://www.ebi.ac.uk/Tools/InterProScan/), except that the transmembrane region of gp210 was predicted with the tool in the TMHMM server v.2.0 (https://www.cbs.dtu.dk/services/TMHMM-2.0/); SMC_prok_A and SMC_prok_B of Tpr/NUA were listed in the TIGR Rice Genome Annotation Resource, and RING-type zinc finger of Elys/ HOS1 was listed in UniProt. Transient and Stable Expression of GFP Fusions in Arabidopsis Genomic DNAs and cDNAs of all 30 Arabidopsis nucleoporins identified were cloned into either pENTR/D-TOPO or pENTR1A (Invitrogen) using specific primers (see Supplemental Table 3 online). To generate the DNA constructs for GFP-tagged nucleoporins, each of the cloned nucleoporin DNAs was transferred from the entry clone to the destination vector pGWB405 (Nakagawa et al., 2007a, 2007b) by an LR reaction. Histone 2B (At5g22880) cDNA was amplified by PCR using gene-specific primers (see Supplemental Table 3 online) and was then cloned into the entry vector pENTR/D-TOPO (Invitrogen). To generate the DNA construct for Histone 2B-tdTomato, the cloned cDNA was transferred from the entry vector to destination vector pGWtd by an LR reaction. Protoplasts derived from cultured Arabidopsis cells were transiently transformed with constructs encoding GFP-tagged nucleoporins using the polyethylene glycol method (Tamura et al., 2003). Arabidopsis plants and tobacco BY-2 cells were transformed by infection with Agrobacterium tumefaciens. The cells were inspected by confocal laser scanning microscopy and differential interference contrast microscopy (model LSM510 META; Carl Zeiss). RT-PCR Analysis Total RNA was isolated from 10-d-old seedlings using a RNeasy Plant Mini kit (Qiagen). Reverse transcription was performed using a kit (Ready- To-Go RT-PCR beads; GE Healthcare) with an oligo(dT)12-18 primer. Gene-specific primers are given in Supplemental Table 3 online. ACT2 was amplified in 27 PCR cycles and NUP136 in 30. PCR products were visualized with ethidium bromide. In Vitro Pollen Germination Assay An in vitro pollen germination assay was performed as described previously (Boavida and McCormick, 2007). Pollen grains were inspected by light microscopy 20 h after germination. Confocal Laser ScanningMicroscopy Fluorescence confocal images were obtained using a laser scanning microscope (Zeiss LSM510 META; Carl Zeiss) equipped with the 405-nm line of a blue diode laser, 488-nm line of a 40-mWAr/Kr laser, or the 544- nm line of a 1-mW He/Ne laser and a 3100 1.45–numerical aperture (NA) oil immersion objective (alpha Plan-Fluar, 000000-1084-514; Carl Zeiss), 363 1.2-NA water immersion objective (C-Apochromat, 441777-9970- 000; Carl Zeiss), or 340 0.95-NA dry objective (Plan-Apochromat, 440654-9902-000; Carl Zeiss). Image analysis was performed using LSM image examiner software (Carl Zeiss). The data were exported as 8-bit TIFF files and processed using either Adobe Photoshop Elements 4.0 (Adobe Systems) or ImageJ 1.40g (National Institutes of Health). FRAP Experiment FRAP experiments were performed with a confocal laser scanning microscope (LSM510; Carl Zeiss) using a 340 0.95-NA dry objective (Plan-Apochromat, 440654-9902-000; Carl Zeiss) and a completely open pinhole. Fluorescence on the particular region (22 3 22 pixels) of nuclear envelope was bleached with full laser power. After photobleaching, images were acquired for 790 s at 30-s intervals. SDS-PAGE and Immunoblot Analysis Protein extracts from seedlings and isolated nuclei were subjected to SDS-PAGE followed by either Coomassie Brilliant Blue staining or immunoblot analysis. Immunoreactive signals were detected with the ECL detection system (GE Healthcare) using anti-GFP antibody (JL-8; Clontech) (1:3000). Antibodies were diluted with Solution 1 of an immunostaining kit (Can Get Signal Immunoreaction Enhancer Solution; Toyobo). Immunoreactive signals were detected with the ECL detection system (GE Healthcare). Immunoprecipitation Immunoprecipitation was performed with mMACS Epitope Tag Protein Isolation Kits (Miltenyi Biotec). Whole seedlings of each transgenic Arabidopsis plant (;0.5 g) expressing GFP-tagged nucleoporins were homogenized on ice in 2 mL of buffer containing 50 mM HEPES-KOH, pH 7.5, 0.15 M NaCl, 0.5% (v/v) Triton X-100, and 0.1% (v/v) Tween 20. Homogenates were centrifuged at 10,000g for 15 min at 48C to remove cellular debris. The supernatants were mixed with magnetic beads conjugated to an anti-GFP antibody (Miltenyi Biotec) and then incubated on ice for 10 min. The mixtures were applied to m Columns (Miltenyi Biotec) in a magnetic field to capture the magnetic antigen-antibody complex. After extensive washing with the buffer, immunoaffinity complexes were eluted with 50 mL of either 0.1 M Na2CO3 solution, pH 11.0, or 23 SDS sample buffer containing 100 mM Tris-HCl, pH 6.8, 4% (w/v) SDS, 20% (w/v) glycerol, and 5%(v/v) 2-mercaptoethanol. Fractions elutedwith 0.1MNa2CO3 were subsequently neutralized with 5 mL of 1MMES. Peptide Preparation for Tandem Mass Spectrometry Analysis The immunoprecipitates were reduced-alkylated and then treated with 0.01 mg/mL trypsin (sequence grade; Promega) in 50 mM ammoniumbicarbonate and incubated at 378C for 16 h. The digested peptides were recovered twice with 20 mL of 5% (v/v) formic acid in 50% (v/v) acetonitrile. The extracted peptides were combined and then evaporated to 10 mL in a vacuum concentrator. For in-gel digestion, the protein components of the immunoprecipitates were separated on a 3-cm-long SDS gel. The gel slice isolated from each lane was cut into three fractions according to molecular mass: a <50-kD fraction, a 50- to 100-kD fraction, and a >100-kD fraction. Each excised gel fraction was treated twice with 25mMammonium bicarbonate in 30% (v/v) acetonitrile for 10 min followed by 100% (v/v) acetonitrile for 15 min, and then dried in a vacuum concentrator. The dried gel was treated with 0.01 mg/mL trypsin in 50 mM ammonium bicarbonate and incubated at 378C for 16 h. The digested peptides were recovered twice with 20 mL of 5%(v/v) formic acid in 50% (v/v) acetonitrile. The extracted peptideswere combined and then evaporated to 10 mL in a vacuum concentrator. Mass Spectrometric Analysis and Database Search Liquid chromatography–tandem mass spectrometry (MS/MS) analyses were performed using the LTQ-Orbitrap XL-HTC-PAL system. Trypsin digests were loaded on the column (75-mm internal diameter, 15-cm length; L-Column, CERI) using the Paradigm MS4 HPLC pump (Michrom BioResources) and HTC-PAL Autosampler (CTC Analytics), and were eluted by a gradient of 5 to 45% (v/v) acetonitrile in 0.1% (v/v) formic acid for 70 min. The eluted peptides were introduced directly into an LTQOrbitrap XL mass spectrometer (Thermo) with a flow rate of 300 nL/min and a spray voltage of 2.0 kV. The range of MS scan was m/z 450 to 1500. The top three peaks were subjected to MS/MS analysis. MS/MS spectra were analyzed by the Mascot server (version 2.2) in house (Perkins et al., 1999) (https://www.matrixscience.com/) and compared against proteins registered in TAIR8. The Mascot search parameters were set as follows: threshold of the ion score cutoff, 0.05; peptide tolerance, 10 ppm; MS/MS tolerance,60.8 D; and peptide charge, 2+ or 3+. The searchwas also set to allowone missed cleavage by trypsin, a carboxymethylationmodification of Cys residues, and a variable oxidation modification of Met residues. In Situ Hybridization The poly (A)+ RNA in situ hybridization was performed as previously described (Gong et al., 2005). The 45-mer oligo(dT) labeled with one fluorescein molecule at the 59-end (Hokkaido system science, Hokkaido, Japan) was used as a probe. The stained cells were inspected with a confocal laser scanning microscope (Carl Zeiss). Hoechst Staining The nuclei in rosette leaves from 2-week-old plants were stained for 30 min with 1 mg/mL Hoechst 33342 solution, 3.7% (w/v) paraformaldehyde, 10% (v/v) DMSO, 3% (v/v) Nonidet P-40, 50 mM PIPES-KOH, pH 7.0, 1mM MgSO4, and 5mMEGTA. The stained nuclei were inspected with a confocal laser scanning microscope by exciting with a 405-nm diode laser (Carl Zeiss). Accession Numbers Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the accession numbers shown in Table 1. Supplemental Data The following materials are available in the online version of this article. Supplemental Figure 1. Fluorescence Images of Protoplasts Derived from Cultured Cells Transiently Expressing GFP-Tagged Nucleoporins. 12 of 14 The Plant Cell Supplemental Figure 2. Identification of Arabidopsis Nup43. Supplemental Figure 3. Identification of Arabidopsis Elys/HOS1. Supplemental Figure 4. The Pollen in nup136/nup1 Mutants Exhibits a Low Germination Rate. Supplemental Figure 5. Abnormal Accumulation of Poly(A)+ RNA in the Nuclei of nup136 Mutants. Supplemental Table 1. Eleven Nucleoporins Identified by Mass Spectrometry of Immunoprecipitates from Transgenic Arabidopsis Plants Expressing Nup93a-GFP. Supplemental Table 2. Information for Domain Positions Used in Figure 4. Supplemental Table 3. Primers Used in This Study. Supplemental Data Set 1. Proteins Identified by Mass Spectrometry of Immunoprecipitates from RAE1-GFP Transgenic Plants. Supplemental Data Set 2. Proteins Identified by Mass Spectrometry of Nup93a-GFP Immunoprecipitates. Supplemental Data Set 3. Proteins Identified by Mass Spectrometry of Nup43-GFP Immunoprecipitates. Supplemental Data Set 4. Proteins Identified by Mass Spectrometry of Nup50a-GFP Immunoprecipitates. Supplemental Data Set 5. Proteins Identified by Mass Spectrometry of Nup136-GFP Immunoprecipitates. |
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