Yeast two‐hybrid (Y2H) assay was performed using the Y2H Gold‐Gal4 system (Clontech, http://www.clontech.com ). Full‐length CDS, the containing domains (MATE1, MATE2 and the mutant MATE1gfd1) of GFD1, and the sugar transporters CDS (OsSUT1‐5, OsSWEET4, OsSWEET11 and OsSWEET15), were cloned into pGADT7 (AD) and pGBKT7 (BD), respectively. Yeast transformations were completed following the manufacturer's instructions (Clontech) and cultured on SD/‐Trp‐Leu or SD/‐Trp‐Leu‐His‐Ade medium containing X‐ɑ‐gal at 30°C in the dark for about 3 days. Primers used are given in Table S1 .
Y2h gold gal4 system
Manufactured by Takara Bio
Sourced in United States
The Y2H Gold-Gal4 system is a yeast two-hybrid (Y2H) assay kit developed by Takara Bio. It is designed to study protein-protein interactions by detecting the activation of reporter genes. The system utilizes the Gal4 transcription factor, which is split into a DNA-binding domain and an activation domain, allowing for the monitoring of interacting proteins.
Automatically generated - may contain errors
10 protocols using y2h gold gal4 system
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Yeast Two-Hybrid Assay for Protein-Protein Interactions
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Yeast Two-Hybrid Assay for MAPK Interactions
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Yeast two-hybrid assays were performed with the Y2H Gold-Gal4 system (Clontech). DsMEK1-X1/2 were inserted into the pGADT7 vectors, and DsMAPK1 (NCBI, GenBank: EF186770.1) and DsMAPKKK genes were inserted into the pGBKT7 vectors to form the bait and prey constructs (primers No. 7–44 in Table S1). The bait and prey constructs were transformed into yeast strain Y2H Gold according to the manufacturer’s instructions (Clontech). The yeast cells were cultured on SD/-Trp/-Leu/-His/-Ade medium containing X-ɑ-gal at 28 °C in the dark for three days.
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Yeast Two-Hybrid Analysis of OsCDC48 Interactions
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Yeast two-hybrid assays were performed with the Y2H Gold-Gal4 system (Clontech, http://www.clontech.com ). DNA fragments containing the full coding sequences of OsCDC48, OsCDC48E and OsCDC48–psd128 genes were inserted into the pGBKT7 and pGADT7 vectors to from the bait and prey constructs, respectively. The bait and prey constructs were transformed into yeast strain Y2H Gold according to the manufacturer’s instruction (Clontech, http://www.clontech.com ). The yeast cells were cultured on SD/-Trp-Leu or SD/-Trp-Leu-His-Ade medium containing X-ɑ-gal and Aureobasidin A (AbA) at 30 °C in darkness for 3 days. The pull-down assay was conducted as follows: 50 µL equilibrated Glutathione High Capacity Magnetic Agarose Beads (Sigma, St Louis, USA) was mixed with 500 µg of each recombinant protein in 600 µL pull-down buffer (50 Mm Tris–HCl pH = 7.5, 5% glycerol, 1 mM EDTA, 1 mM DTT, 1 Mm PMSF, 0.01% Nonidet P-40, 150 mM KCl) under 4 °C for 2 h. The bound proteins together with the beads were collected by a magnetic shelf (Invitrogen, Carlsbad, USA), washed with pull-down buffer twice, eluted with 50 µL 1 × PBS and immune detected by GST (Cat: CW0085, CWBIO, Beijing, China), HIS (Cat: CW0083, CWBIO, Beijing, China) antibodies respectively.
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Yeast Two-Hybrid Screening of OsMPK6 Interactors
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The Y2H Gold-Gal4 system (Clontech) was used to perform yeast twohybrid assays. The full-length coding sequences of OsMPK6 and DST were inserted into the pGBKT7 and pGADT7 vectors to prepare the bait and prey constructs, respectively, which were transformed into yeast strain Y2H Gold. For yeast two-hybrid screening of OsMPK6 interacting proteins, the yeast cells were cultured on SD/-Trp medium, and 500-mL cultures were grown to prepare competent cells for transformation with the pGADT7 plasmid library as described by Chen et al. (2019) . After transformation, yeast cells containing the pGADT7 plasmid library were cultured on SD/-Trp-Leu-His or SD/-Trp-Leu-His-Ade medium containing X-ɑ-gal at 30°C in the dark for 3 d, after which the positive colonies were selected, and the inserts in the pGBKT7 vectors were sequenced. For the specific yeast twohybrid assays, the bait and prey constructs were co-transformed into yeast strain Y2H Gold according to the manufacturer's instructions (Clontech). The yeast cells were cultured on different media for observation. SD/-Trp is yeast culture medium without Trp. SD/-Trp-Leu-His is yeast culture medium without Trp, Leu, and His. SD/-Trp-Leu-His-Ade is culture medium without Trp, Leu, His, and Ade. The PCR primers used for the yeast twohybrid assays are listed in the Supplemental Data Set.
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Yeast Two-Hybrid Assay for Protein-Protein Interactions
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Yeast two‐hybrid assay was performed with the Y2H Gold‐Gal4 system (Clontech, USA). The CDS of DGW1 was introduced into the pGBKT7 vector, and full‐length cDNA of OsUBP1a and OsUBP1b were cloned into the pGADT7 vector. The constructs were co‐transformed into Y2H Gold cells according to the manufacturer's instructions (Clontech, USA). Interactions were detected on the SD/−Trp‐Leu‐His‐Ade medium.
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In Vivo Protein-Protein Interaction Assays
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To detect in vivo protein-protein interactions, specific yeast two-hybrid assays were performed using the Y2H Gold-Gal4 system (Clontech). In brief, the bait and prey constructs were prepared ahead of time. DNA fragments containing the full or truncated OsHAL3 and D3 coding sequences were inserted separately into the pGBKT7 and pGADT7 vectors, respectively. The corresponding bait and prey vectors were then co-transformed into yeast strain Y2HGold and cultured on an SD drop-out medium containing X-α-gal at 30°C in the dark. Transformants were grown on plates until a blue color developed. Detailed experimental procedures were performed according to the manufacturer's instructions (Clontech). The PCR primers used in the yeast two-hybrid assays are given in Supplemental Table 4.
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Yeast Two-Hybrid Assay and Transformation of Dunaliella salina
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Yeast two-hybrid assays were performed with the Y2H Gold-Gal4 system (Clontech). DsMEK1-X1/2 were inserted into the pGADT7 vectors, and DsMAPK1 (NCBI, GenBank: EF186770.1) and DsMAPKKK genes were inserted into the pGBKT7 vectors to form the bait and prey constructs (primers No. 7-44 in Table S1). The bait and prey constructs were transformed into yeast strain Y2H Gold according to the manufacturer's instructions (Clontech). The yeast cells were cultured on SD/-Trp/-Leu/-His/-Ade medium containing X--gal at 28 ˚C in the dark for three days.
Construction of DsMEK1-X1/X2 overexpression strains and DsMEK1-X2 knockdown strains
The pGreen-0029 binary vector was modi ed and used as the D. salina transformation vector. To generate the DsMEK1 transformation constructs, a 573 bp chloramphenicol resistance gene (Cmr) was ampli ed and inserted into pGreen-0029 as a dominant selectable marker (primers No. 45-46 in Table S1). To construct pGreen-0029-Cmr-DsMEK1 overexpression strains (DsMEK1-X1-oe and DsMEK1-X2-oe), the fulllength CDS of DsMEK1-X1 and DsMEK1-X2 were ampli ed by PCR (primers No. 47-48 in Table S1) and then inserted into the modi ed pGreen-0029-Cmr vector. To generate the DsMEK1-X2 knockdown construct DsMEK1-X2-RNAi strains, interference sequences were obtained by chemical synthesis (Sangon Biotech Co., Ltd) and inserted into the modi ed pGreen-0029-Cmr vector.
Construction of DsMEK1-X1/X2 overexpression strains and DsMEK1-X2 knockdown strains
The pGreen-0029 binary vector was modi ed and used as the D. salina transformation vector. To generate the DsMEK1 transformation constructs, a 573 bp chloramphenicol resistance gene (Cmr) was ampli ed and inserted into pGreen-0029 as a dominant selectable marker (primers No. 45-46 in Table S1). To construct pGreen-0029-Cmr-DsMEK1 overexpression strains (DsMEK1-X1-oe and DsMEK1-X2-oe), the fulllength CDS of DsMEK1-X1 and DsMEK1-X2 were ampli ed by PCR (primers No. 47-48 in Table S1) and then inserted into the modi ed pGreen-0029-Cmr vector. To generate the DsMEK1-X2 knockdown construct DsMEK1-X2-RNAi strains, interference sequences were obtained by chemical synthesis (Sangon Biotech Co., Ltd) and inserted into the modi ed pGreen-0029-Cmr vector.
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Yeast Two-Hybrid Screening and Genetic Manipulation of DsMEK1
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Yeast two-hybrid assays were performed with the Y2H Gold-Gal4 system (Clontech). DsMEK1-X1/2 were inserted into the pGADT7 vectors and DsMAPK1 (NCBI, GenBank: EF186770.1), DsMAPKKK genes were inserted into the pGBKT7 vectors to form the bait and prey constructs (primers No. 5-43 in Table S1).
The bait and prey constructs were transformed into yeast strain Y2H Gold according to the manufacturer's instructions (Clontech). The yeast cells were cultured on the SD/-Trp/-Leu/-His/-Ade medium containing X--gal at 28 ˚ C in the dark for three days.
Construction of DsMEK1-X1/X2 overexpression strains and DsMEK1-X2 knock down strains
The pGreen-0029 binary vector was modi ed and used as the D. salina transformation vector. To generate the DsMEK1s transformation constructs, a 573 bp chloramphenicol resistance gene (Cmr) was ampli ed and inserted to the pGreen-0029 as a dominant selectable marker (primers No. 44-45 in Table S1). To construct pGreen-0029-Cmr-DsMEK1 overexpression strains (DsMEK1-X1-oe and DsMEK1-X2-oe), the full-length CDS of DsMEK1-X1 and DsMEK1-X2 were ampli ed by PCR (primers No. 46-47 in Table S1), then inserted them into the modi ed pGreen-0029-Cmr vector. To generate the DsMEK1-X2 knockdown construct DsMEK1-X2-RNAi strains, obtaining interference sequences by chemical synthesis (Sangon Biotech Co., Ltd), and inserted into the modi ed pGreen-0029-Cmr vector.
The bait and prey constructs were transformed into yeast strain Y2H Gold according to the manufacturer's instructions (Clontech). The yeast cells were cultured on the SD/-Trp/-Leu/-His/-Ade medium containing X--gal at 28 ˚ C in the dark for three days.
Construction of DsMEK1-X1/X2 overexpression strains and DsMEK1-X2 knock down strains
The pGreen-0029 binary vector was modi ed and used as the D. salina transformation vector. To generate the DsMEK1s transformation constructs, a 573 bp chloramphenicol resistance gene (Cmr) was ampli ed and inserted to the pGreen-0029 as a dominant selectable marker (primers No. 44-45 in Table S1). To construct pGreen-0029-Cmr-DsMEK1 overexpression strains (DsMEK1-X1-oe and DsMEK1-X2-oe), the full-length CDS of DsMEK1-X1 and DsMEK1-X2 were ampli ed by PCR (primers No. 46-47 in Table S1), then inserted them into the modi ed pGreen-0029-Cmr vector. To generate the DsMEK1-X2 knockdown construct DsMEK1-X2-RNAi strains, obtaining interference sequences by chemical synthesis (Sangon Biotech Co., Ltd), and inserted into the modi ed pGreen-0029-Cmr vector.
9
Protein Interactions Revealed via Yeast Two-Hybrid
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Yeast two-hybrid assays were performed with the Y2H Gold-Gal4 system (Clontech). The DNA fragments containing the AET1, RACK1A, and eIF3h coding sequences were cloned separately into the pGBKT7 and pGADT7 vectors to form the bait and prey constructs, respectively. For all pGBKT7 constructs the EcoRI and SalI sites were used, while for all pGADT7 constructs the EcoRI and BamHI sites were used. All bait and prey constructs were transformed into the yeast Y2H Gold strain as directed by the manufacturer. The yeast cells were cultured on SD/-Trp-Leu-His or SD/-Trp-Leu-His-Ade medium containing X-a-gal at 30 C for 3 days. In the self-activation assays, the SD/-Trp-Leu-His culture medium also contained 5 mM 3-AT. Three biological replicates were performed for all yeast cell dilutions with the same results, and the figures show results for only one experiment. The PCR primers used for the yeast two-hybrid assays are listed in Supplemental Table 2.
10
Yeast Two-Hybrid Screening of Seedling and Spikelet cDNA Libraries
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Yeast two-hybrid screening assays were performed using the Y2H Gold-Gal4 system (Clontech). Our laboratory had previously constructed two yeast plasmid prey libraries, one from mixed seedlings and the other from mixed spikelets. The libraries were both plasmid based. We cloned the full-length cDNAs into pGBKT7 to form the prey plasmid and then transformed the plasmid into the Y2H Gold strain. The yeast cells were cultured on SD/-Trp medium, and 500-ml cultures were grown to prepare competent cells to transform with the pGADT7 plasmid libraries. After transformation, the yeast cells containing the pGADT7 plasmid libraries were cultured on SD/-Trp-Leu-His or SD/-Trp-Leu-His-Ade medium containing X-a-gal at 30 C for 3 days. SD/-Trp-Leu-His is yeast culture SD medium without tryptophan, leucine, and histidine, and SD/-Trp-Leu-His-Ade is SD medium lacking tryptophan, leucine, histidine, and adenine. The colonies were selected, and the inserts in the pGBKT7 vectors were sequenced. Screening was performed following the manufacturer's (Clontech) instructions.
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