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Acta Agron Sin ›› 2009, Vol. 35 ›› Issue (7): 1253-1360.doi: 10.3724/SP.J.1006.2009.01253

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Codl Tolerance of Transplastomic Tobacco Lines Carrying Insect Antifreeze Protein

WANG Yan,MA Ji*,HUANG Wei,QIU Li-Ming,YE Feng,ZHANG Fu-Chun   

  1. Xinjiang Key Laboratory of Biological Resources and Genetic Engineering,College of Life Science and Technoogy,Xinjiang University,Urumqi 830046,China
  • Received:2008-09-05 Revised:2009-02-18 Online:2009-07-12 Published:2009-05-19
  • Contact: MA Ji, E-mail: majiuci@xju.edu.cn; Tel: 0991-8583259
  • About author:bluelovewy@126.com

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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 analysis showed that the MpAFP149 gene was successfully integrated into the tobacco chloroplast genome, but the transgenicplants 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.

Key words: Antifreeze protein gene MpAFP149, Chloroplast transformed vector, Tobacco, Cold tolerance


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