Abstract: Most of crops are susceptible to cold and frost. When temperature decreases to -1℃, ice makes plant cells desiccated and cellular membrane shrinked. Expression of insect antifreeze protein genes with very high thermal hysterisis activity in plants is a possible way for increasing the cold tolerance of cold-sensitive plants. The MPAFP149 gene from Xinjiang desert insect Microdera punctipennis dzhunarica was constructed to the plant expression vector pCAMBIA1302 by sub-cloning, and then the recombined vector pCAMBIA1302-MPAFP149 was transformed into Agrobacterium tumefacines EHA105. Transgenic tobacco was obtained via leaf-disc method by Agrobacterium-mediated transformation. PCR and PCR-Southern analysis showed that the MPAFP149 gene was successfully integrated into the tobacco genome. The result of RT-PCR also verified that MPAFP149 gene was transcripted at mRNA level. The relative conductivity of T0 generation of transgenic tobacco at －1℃ for 48 h was 28.83%, significantly lower than that of wild tobacco which was 82.91%. Meanwhile, the phenotype of transgenic tobacco was superior to that of wild tobacco, suggesting that transgenic tobacco had better cold tolerance than wild tobacco. Recovering experiment at the room temperature indicated that transgenic tobacco was able to recover from cold stress and regenerate. The results proved that transgenic tobacco carrying MPAFP149 gene was more cold tolerant than wild tobacco, which provided a theoretical and applied foundation for relieving cold damage to cold-sensitive crops in spring.

Key words: Antifreeze proteins, Plant expression vector, Tobacco, Cold tolerance