Five OsMYB61 bait fragments of pMYB61–1 (− 1946, − 1258), pMYB61–2 (− 1607, − 1258), pMYB61–3 (− 1258, − 897), pMYB61–4 (− 870, − 356) and pMYB61–5 (− 356, − 1) were cloned into the pHIS2 vector between EcoRI and SacI sites and integrated into the genome of yeast strain Y187 (MATα, ura3–52, his3–200, ade2–101, trp1–901, leu2–3, 112, gal4∆, met−, gal80∆, URA3:: GAL1UAS-GAL1TATA-lacZ, MEL1). For the self-activation test, promoter bait strains were grown on the SD/−Trp, -His (a synthetic Trp and His dropout medium) media in the presence of 0 mM, 10 mM, 30 mM and 50 mM 3-aminotriazole (3-AT). We performed the yeast one-hybrid screening using the BD Matchmaker One-hybrid Library Construction and Screening Kit (K1617–1, Clontech) according to the user manual (PT3529–1, Clontech). The cDNA library of the internodes tissue was constructed with the pGADT7-Rec2 vector (Clontech). The promoter bait strains were then mated with the “pGADT7-Rec2-cDNA” library and screened on the SD/−Leu -Trp -His selection media containing 30 mM 3-AT. Positive colonies were selected for yeast plasmid isolation or PCR with primers AD-F and AD-R. The PCR was performed according to the following program, 95 °C 5 min, 95 °C 30 s, 56 °C 30 s, 72 °C 2 min, 36 cycles, 72 °C 10 min, 12 °C pause.
Pgadt7 rec2 vector
Manufactured by Takara Bio
The PGADT7‐Rec2 vector is a plasmid used in molecular biology research. It enables the expression of recombinant proteins in yeast cells. The vector contains a yeast promoter, a multiple cloning site for inserting genes of interest, and a selectable marker for identifying transformed yeast cells.
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5 protocols using pgadt7 rec2 vector
1
Yeast One-Hybrid Screening of OsMYB61 Promoters
2
Yeast One-Hybrid Assay for PagWOX11/12a Transcriptional Regulation
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The full‐length coding sequence (CDS) of PagWOX11/12a was inserted into the pGADT7‐Rec2 vector (Clontech) to generate the effector construct pGAD‐PagWOX11/12a. Three tandem copies of the WOX11‐binding motif ‘TTAATGC’ and the PagCYP736A12 promoter fragment upstream of the transcription starting sites (spanned from −1 to −1000) were PCR amplified and inserted into the pHIS2 vector (Clontech) to generate recombinant reporter constructs. The effector and reporter constructs were co‐transformed into yeast Y187 strain cells using a Matchmaker™ Yeast One‐Hybrid System (Clontech) and then placed on SD/−Leu/−Trp (DDO) and SD−Trp/−Leu/−His (TDO) media containing 30 mm 3‐AT. The Y1H assay was conducted as previously described (Wang et al., 2020a (link)). The pGAD‐PagWOX11/12a and p53HIS2 were used as negative control. The pGAD‐Rec2‐53 and p53HIS2 were used as positive control. The experiments were performed in triplicates. All primers used are shown in Table S1 .
3
Cloning and Yeast Expression of CsTu
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The coding region sequences of CsTu were cloned into the pGADT7‐rec2 vector (Clontech). The promoter of CsTS1 in 3546‐1 and 3546‐2 was cloned into the pHIS2 vectors using the primers listed in Table S6 . All recombinant constructs were separately transformed into the yeast strain Y187. Transformants were grown on SD media ‐His/‐Leu/‐Trp, but containing X‐gal to observe the colour development of yeast colonies.
4
Yeast-one-hybrid assay for BpGRF3 transcription
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The coding sequence of BpGRF3 was cloned into the pGADT7‐Rec2 vector (Clontech) to generate BpGRF3–pGADT7‐Rec2 constructs. The ABRE in the BpGRP1 promoter was inserted into the pAbAi vector (Clontech) immediately upstream of the AUR1‐C gene. These constructs were cotransformed into the Saccharomyces cerevisiae strain Y187, and the Y1H assay was conducted as previously described (Liu et al., 2020 (link)). The primers are listed in Table S1 .
5
Identifying miR528 promoter regulators
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The miR528 promoter region containing the GTAC motifs was cloned into the pHis2 vector (Clontech) to generate the reporter construct miR528pro:His. The GTAC motifs in the miR528 promoter were also mutated into GATC to generate a mutant miR528 promoter, which was cloned into the pHis2 vector to generate the reporter construct miR528promut:His. To generate the AD-SPLs, the full-length coding sequences of SPL1-19 were individually amplified using PCR and cloned into the pGADT7Rec2 vector (Clontech), which contains Leu and Trp biosynthesis genes (Takara Bio). The individual AD-SPLs were separately co-transformed into the yeast strain Y187 alongside the reporter construct. The transformants were then grown on SD/-Trp-Leu and SD/-Trp-Leu-His dropout medium for selection or the activation test. The primers used are listed in Supplemental Data 2.
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