Bstbi restriction endonuclease

The expression vector of proMdFLS1-AbAi was constructed using proMdFLS1 as the bait sequence. The CDS region of MdSCL8 was cloned into pGADT7 vector to form PGADT7-MdSCL8. The proMdFLS1-AbAi plasmid linearized by BstBI restriction endonuclease (NEB, Beijing, China) was transformed into yeast competence to obtain bait yeast [proMdFLS1-AbAi]. The library screening experiment was conducted according to the yeast one-hybrid instruction of Shanghai OE Biotech Co., Ltd. (Shanghai, China). The screened genes were subjected to yeast colony PCR, sequencing analysis, and point-to-point verification.

The DNA sequence of the Fshb gene was inserted into the pAbAi vector. The recombinant plasmid pAbAi-Fshb was linearized using BstBI restriction endonuclease (R0519, NEB, USA). The enzyme cleavage system is detailed in the additional file (Additional file 2: Table S4). Integration of pAbAi-Fshb into the Y1Hgold yeast genome after successful linearization was confirmed by PCR. A self-activation assay of Y1Hgold [pAbAi-Fshb] was performed after successful integration by PCR. The FOXP2 overexpression plasmid was transferred into Y1Hgold[pAbAi-Fshb] in the absence of self-activation or with slight self-activation. The interactions between FOXP2 and Fshb DNA were explored by point-to-point validation and point-plate validation experiments.