Cloning And Antifreezing Analysis Of PicW Homologous Genes In Pine Trees

Low temperature is one of the important abiotic stress, seriously affecting the growth and distribution range of plants. It is of great significance to improve plant varieties and promote the plant variety better adapted to low temperature environment with exploring the antifreeze molecμlar mechanisms of plants and detecting antifreeze related genes. Based on the research of the early stage of our team, PicW homologous genes PmCAP and LpCAP were cloned from two cμltivars of pine-Pseudotsuga menziesii and Larix principis-rupprechtii Mayr in conifers (Pinaceae), and their identity with PicW were 77% and 71% respectively. The complete length of PmCAP CDS (coding sequence) was 576 bp, encoding 191 amino acids with a 5.39 isoelectric point. And the complete length of LpCAP CDS (coding sequence) was 687 bp encoding 228 amino acids with a 5.44 isoelectric point. The specific K pieces repeat unit of dehydrin protein was showed in all of protein PmCAP, LpCAP and PicW, and a lot of hydrophobic amino acids such as Lys, Ala, Thr, etc. were contained in these proteins. Secondary structure was mainly composed of alpha helix and random coil, which contributed to the stability of the protein space structure. And then transfered genes PmCAP and LpCAP into E.coli, soluble objective protein expression by IPTG induced. Strains cultivated at a temperature of -5℃, the lethal dose 50%(LD-50) time for the transgenic strains were between 50h-54h, but for the controls was 32h; while strains cμltivated at -20℃, the LD-50 time for the transgenic strains was between 38h-41h, but for the controls was 25 h, which showed the antifreeze property of LaCAP was higher than PmCAP. Furthermore, extracted and purified these antifreeze proteins. A Differential Scanning Calorimeter determination shows a characteristic thermal hysteresis over 0.27℃ and 0.72℃ respectively, revealing significant higher than a temperature of 0.05 ℃ of the control group BSA. Resμlts of the research indicate that both of the cloned genes possess good antifreeze property, which suggested both of them can be used as resources for genetic engineering of plant cold tolerance.