Cloning of an antifreeze protein gene from carrot and its influence on cold tolerance in transgenic tobacco plants

The gene encoding a 1,099-bp carrot antifreeze protein (AFP) was amplified by the polymerase chain reaction (PCR) using genomic DNA from carrot seedlings as the template. Sequencing results indicated a difference of three bases between this cloned gene and that published in GenBank. The cloned antifreeze protein gene was expressed in Escherichia coli, and a fusion protein of about 60 kDa was detected after isopropyl thiogalactoside induction. This AFP gene was also cloned into binary vector pCAMBIA2300 with the CaMV 35S promoter and used to transform tobacco NC82. PCR and Southern blot results verified integration of this gene into the genome of tobacco and reverse transcription-PCR verified that this gene had been expressed in transgenic tobacco. Experiments confirmed that transgenic tobacco plants displayed greater stamina than wild-type ones when subjected to cold treatment. The genetic stability of the transgenic lines was analyzed, and findings confirmed that transgenic expression of the carrot AFP gene could enhance the tolerance of plants to cold or frigid conditions.