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Expression of a Carrot antifreeze protein gene in Escherichia coli

Forestry Studies in China 2003年第4期5卷 22-25页

作者： Ma Xinyu Shen Xin Lu Cunfu College of Biological Sciences and Biotechnology Beijing Forestry UniversityBeijing 100083P.R.China

The recombinant expression vector pET43.1b-AFP, which contains full encoding region of a carrot 36 kD antifreeze protein (AFP) gene was constructed. The recombinant was transformed into expression host carrying T7 RNA polymerase gene (DE3 lysogen) and induced by 1 mmol稬-1 IPTG (isopropyl--D-thiogalactoside) to express 110 kD polypeptide of AFP fusion protein. The analysis of product solubility revealed that pET43.1b-AFP was predominately soluble, and the expressed amount reached the maximum after the IPTG treatment for 3 h.

关键词： antifreeze protein (AFP) fusion protein induced expression polymerase chain reaction antifreeze protein gene

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Expression of a Carrot 36 kD antifreeze protein gene Improves ColdStress Tolerance in Transgenic Tobacco

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Forestry Studies in China 2005年第4期7卷 11-15页

作者： Xu Wen-li Liu Mei-qin Shen Xin Lu Cun-fu College of Biological Sciences and Biotechnology Beijing Forestry University Beijing 100083 P. R. China

antifreeze proteins （AFPs） enable organisms to survive under cold conditions, and have great potential in improving cold tolerance of cold-sensitive plants, In order to determine whether expression of the carrot 36 kD antifreeze protein gene confers improved cold-resistant properties to plant tissues, we tried to obtain transgenic tobacco plants which expressed the antifreeze protein. Cold, salt, and drought induced promoter Prd29A was cloned using PCR from Arabidopsis. Two plant expression vectors based on pBI121 were constructed with CaMV35S：AFP and Prd29A：AFP. Tobacco plantlets were transformed by Agrobacterium-medicated transformation. PCR and Southern blotting demonstrated that the carrot 36 kD afp gene was successfully integrated into the genomes of transformed plantlets. The expression of the afp gene in transgenic plants led to improved tolerance to cold stress. However, the use of the strong constitutive 35S cauliflower mosaic virus （CaMV） promoter to drive expression of afp also resulted in growth retardation under normal growing conditions. In contrast, the expression of afp driven by the stress-inducible Prd29A promoter from Arabidopsis gave rise to minimal effects on plant growth while providing an increased tolerance to cold stress condition （2℃）. The results demonstrated the prospect of using Prd29A-AFP transgenic plants in cold-stressed conditions that will in turn benefit agriculture.

关键词： antifreeze protein gene stress inducible-promoter tobacco genetic transformation cold tolerance

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