Cold Tolerance of Transplastomic Tobacco Lines Carrying Insect Antifreeze Protein

Abstract

The antifreeze protein gene MpAFP149 from desert insect Microdera punctipennis dzungarica was inserted into soybean chloroplast vector pJY01 to construct recombinant chloroplast vector pJY01-MpAFP149 by designing special primers. The plasmid was then transformed into tobacco by gene gun. Four transplastomic tobacco lines were obtained by spectinomycin screening. PCR and PCR-Southern analyses showed that the MpAFP149 gene was successfully integrated into the tobacco chloro-plast genome, but the transgenic plants exhibited low homoplasmy. The result of RT-PCR also validated that MpAFP149 gene was transcribed at mRNA level. The antifreeze effect of transplastomic tobacco with insect antifreeze protein gene in its chloroplasts was evaluated by measuring the relative conductivity and comparing the phenotypes of different plants after cold treatment. Wild-type tobacco, transplastomic tobacco and T1 generation of transgenic tobacco containing pCAMBIA1302-MpAFP149 in its nuclei were subjected to –1 ℃ for different days (0, 1, 2, and 3 d). The results showed that transgenic plants with insect antifreeze protein gene in chloroplasts or in nuclei performed better phenotype after cold treatment at –1℃ for three days and recovering at room temperature for five days than wild-type tobacco. After three days, the electrolyte leakage reached 73.7% for wild-type tobacco, 39.2% for chloroplast transgenic plants and 38.2% for nuclei transformed T1 generation tobacco. There was no cold tolerance difference between nuclei transgenic tobacco and heterogeneous tansplastomic tobacco in our research.