Nickel sulfate resin

The CDS of wild-type (wt)/mutated catalase and malate dehydrogenase genes were PCR amplified, digested with restriction enzymes EcoR I/Not I and BamH I/Sal I, and cloned into the prokaryotic expression vector pET-28a (Novagen, Wisconsin, USA). The recombinant His-tagged catalase and malate dehydrogenase proteins were expressed in E. coli BL21 (DE3) and purified using a nickel sulfate resin (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The purity of the purification products was monitored through SDS-PAGE. Briefly, 40 μg of purified proteins was mixed with 6 × SDS-PAGE sampling buffer, denatured by boiling at 100 °C for 5 min, separated by 12% SDS-PAGE, and stained with Coomassie brilliant blue.

Site‐specific mutations of the GhPK6 ORF were generated using the overlap PCR method 17. The full‐length ORF of wild‐type (wt) and mutated GhPK6 were amplified by PCR, digested with BamHI and SacI, and cloned into the prokaryotic expression vector pET‐28a (Novagen, Madison, WI, USA). The primers are listed in Table S1. The recombinant His‐tagged proteins were expressed in the Escherichia coli strain BL21 (DE3) and purified using a nickel sulfate resin following the manufacturer's instructions (Qiagen, Hilden, Germany).