PicW orthologs from spruce with differential freezing tolerance expressed in Escherichia coli

Spruce can grow at an extra low temperature (LT), and is inferred with important antifreezing gene resources. The research here identified 4 different spruce varieties, named as PicW1, PicW2, PicM and PicK. Sequence alignment showed base-substitution and deficiency mutations among them with sequence identity between 97.61% and 99.25%. Each gene was transferred into E. coli, where protein was induced by IPTG (isopropyl-β-d-thiogalactoside). Strains cultured at -5°C showed the lethal dose 50% (LD-50) between 53h and 57h for the transgenic strains, but 35h for the control. Strains cultivated at -20°C showed the LD-50 between 38h and 44h for the transgenic strains, but 25h for the control. Further, the soluble gene proteins were extracted and purified for Differential Scanning Calorimeter (DSC) test, which showed characteristic thermal hysteresis (TH) value of 0.77°C (PicW1), 0.78°C (PicW2), 0.72°C (PicM), and 0.86°C (PicK) respectively, significantly higher than the value of 0.05°C of the control (BSA). Summarily, four homologous proteins showed good antifreeze property with the range from high to low as PicK>PicW2>PicW1>PicM. It suggested that they can be used as resources for genetic engineering of plant cold tolerance.