Pb42ad vector

Effectors were generated by amplifying the coding sequences of TaABFs and TaMYBs from wheat cv. Chinese Spring and independently fusing them with the activation domain of the pB42AD vector (Clontech, Mountain View, CA, USA). The TaPYL‐1B promoter fragments were amplified and cloned into the pLacZi vector (Promega, Madison, WI, USA), driving LacZ reporter expression. The yeast strain EGY48 was created by the co‐transformation of effector and reporter plasmids, and it was then cultured on SD/−Trp/−Ura dropout medium containing X‐gal at 30°C for blue colour development. Empty pB42AD and pLacZi served as negative controls.

The Opaque2 coding sequence was cloned into the pB42AD vector (Clontech, Mountain View, CA) and co-transformed with a modified pLacZ vector (38 (link)) that contained the LacZ reporter driven by the promoter region of ZmRPABC5a or Dek701 in the yeast strain EGY48. The transformants were grown on synthetic defined (SD) medium lacking uracil and tryptophan (SD −Ura −Trp) and containing 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside for blue color development.

For yeast one‐hybrid assay, the coding sequence (CDS) of OsRE1 was amplified by PCR and cloned into the pB42AD vector (Clontech). To generate Pro_Ehd1: LacZ reporter gene, several fragments of Ehd1 promoter were amplified and cloned into the vector pLacZi. Plasmids were co‐transformed into yeast strain EGY48. Transformants were selected and grown on SD/‐Trp‐Ura dropout medium for 72 h, and then transferred onto X‐gal (5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galacto‐pyranoside) plates for blue colour development (Lin et al., 2007).

Y1H assays were performed according to the Yeast Protocols Handbook (Clontech). To generate the GAL4 AD construct as prey, the OsCCA1 coding sequence was amplified by RT-PCR using gene-specific primers (Supplemental Table S1) and cloned into the pGADT7 vector (Clontech) for the CPRG assay and the pB42AD vector (Clontech) for the spotting assay, respectively. As a bait, the promoter fragments from possible OsCCA1 target genes (Ehd1, OsPRR37, and DTH8) were amplified by genomic PCR with gene-specific primers (Supplemental Table S1) and the products were then inserted in the pLacZi vector (Clontech). For the CPRG assay, the yeast YM4271 strain was used as the recipient for bait and prey constructs and β-galactosidase activity was measured by liquid assay using CPRG (Roche Applied Science) according to the Yeast Protocols Handbook (Clontech). For the spotting assay, the EGY48 strain was used as a recipient for bait and prey constructs, and transformants were then grown on SD/-Trp, -Ura +X-gal (80 mg mL^–1) medium for 3 days according to the Yeast Protocols Handbook (Clontech). These experiments were conducted more than three times with independent clones obtained from different yeast transformants.