Studies On The Construction Of Plant Expression Vector Of Phosphatidylethanolamine-binding Family Protein Gene And Its Genetic Transformation

The bad environment is a very important nonbiological force factor influencing agricultural production and ecological environment. In these factors, the low temperature is always an important reason to perplex the agriculture to develop. How to increase the cold resistance of plant and reduce the damage on crops and vegetables is an important research subject in bioscience. With the development of gene engineering technology, It becomes an efficient way to improve the cold resistance of plant that directly increase the cold resistance of plant by transferring anti-cold revelant genes into plant and make them express. The phosphatidylethanolamine-binding family protein gene from Saussurea involucrata Kar.et belongs to anti-cold revelant gene. It is revelant to the stability of cellular membrane, and make an important role in the cold resistance of plant.This study discussed the strategy of constructing the plant expression vector, and firstly constructed the plant expression vector including XLPBP gene. And it established a highly efficient regeneration system of cotton from genes engineering and then transfer the XLPBP gene into cotton, which mediated by Agrobacterium tumefaciens. The main factors influencing regeneration and transformation rate were discussed and the best conduction of regeneration and transformation were determined. The positive regeneration was obtained. The result was summarized as follows:1 construction of the plant expression vector: The fragment of barget gene was amplified by PCR, and the site of EcoRⅠwas added to both ends of the gene. After purified, it was inserted into expression vector pCAMBIA3301 that was digested by EcoRⅠ.2 establishment of a highly efficient regeneration system of cotton: The best explants were stems which were cultured 5 days. The optimum inducing medium is the improved MS +2,4-D 0.1mg/L+Kt 0.1 mg/L, and pH5.8. The stems of B02 were put in Agrobacterium tumefaciens for 10 minutes and then co-cultivated in medium, pH 5.8 for 2 days under no light and the temperature was 22℃.The optimum concentration of kanamycin used in resistant selection was 50 mg/L and cefotaxime used in resistant germ culture was 500mg/L.3 Detection of transforming regeneration cotton: The results of GUS demonstrated the integration of target gene into cotton genome primarily.4 transformation of pollen-tube pathway method: Two types cotton were selected as the acceptor genotype, introduced 100ug/ml DNA at 36 hours after pollination. Many seeds were got. In this study, target gene include non-coding region and pCAMBIA3301 has GUS-bar gene cassette and CaMV 35S promoter. By this, the expression of XLPBP could be improved and the detection of transforming regeneration would be efficient.