Cloneez kit

The genomic DNA was isolated from P. pastoris cells using the TIANamp Yeast DNA Kit (Tiangen, Beijing, China). Based on the genome sequence of P. pastoris GS115, DNA fragments upstream and downstream of the open reading frame of gas1 were amplified from genomic DNA by two pairs of primers (gas1-F1 and gas1-R1, gas1-F2 and gas1-R2, respectively). HIS4, a selectable marker for isolating Pichia recombinant strains, was amplified from plasmid pPIC9 using the primers his4-F and his4-R. The three abovementioned DNA fragments were ligated into a DNA fragment named “GH” via overlap-extension PCR with the primers gas1-LF and gas1-RR. In addition, a DNA fragment designated “OA” containing the origin of replication derived from pBR322 and an ampicillin resistance gene was amplified from plasmid pPIC9 with primers OA-F and OA-R. Finally, the two DNA fragments, GH and OA, were ligated into a circular molecule by homologous recombination using the CloneEZ Kit (GenScript, Nanjing, China), generating the recombination vector pGH01. pGH01 was integrated into the gas1 locus of the P. pastoris chromosome via homologous recombination.

Hormonal reporter constructs pRD29A::LUC, pGH3.3::LUC, pARR6::LUC and pFRK1::LUC were ordered from ABRC (CD3-912, CD3-913, CD3-917 and CD3-919). The remaining reporter constructs were generated by recombination-based cloning (CloneEZ kit, GenScript). The promoter fragments were amplified by PCR from genomic Col-0 DNA (see primer sequences in S1 Table). The plasmid backbone resulted from digesting pFRK1::LUC with BamHI and NcoI. The transfection control plasmid was pAtUBQ10::GUS.