Trehalose Synthase Gene Genetic Transformation Of Tobacco

Drought has been one of the greatest natural disaster threatening agriculture in our country . It affects the growth of plants and sharply reduces the yield. In china, about one-third of the land is in the drought and half-drought state and above 7% of the arable is threatened by drought and salinization. However the number is still increasing with the desertification of land and the lack of fresh water. So, it is an economical and efficient approach for utilizing the drought-stress land better to cultivate a new variety with drought-resistance trait.Trehalose plays a special role in the organism.. It endows the trehalose-producing organisms with the ability to resist drought and freeze because it can protect the structures of the protein and the membrane. Besides, it also has the special protective function on protein products and food.In our experiments, we transformed the Grifola frondos ( bacidiomycete ) trehalose synthase gene (Tsase) into tobacco to make further study to verify the function of the gene. First, a plant expression vector, pBBBT was constructed by inserting Tsase into pBBB-ETR to substitute the ETR gene within the plant expression vector. Then, it was introduced into the super-poison Agrobacterium tumerfaciens EHA105 through the "Triparental mating" method. Twenty regenerated tobacco plantlets were obtained with selection medium under the PPT selective pressure. Three plantlets were selected positive via vitro studies with PPT. We used the method of PCR to detect the trehalose synthase gene in them and there were two PCR-positive plants in the three. Southern blotting analysis suggested that the trehalose synthase gene had been integrated into the genome of the two PCR-positive regenerated plants. Here we used a method of HPLC/ELSD to analyse the content from leaf extract of the two transformants and CK. There were 2.126 mg/gFW and 2.556 mg/gFW trehalose in these twotransformants, respectively, whilst no trehalose was detected in CK. In comparison with CK, obvious morphological variation were observed in the transgenic tobaccos. Their leaves became greener and thicker, chief veins turned bolder and thicker than those of CK. In addition, drought-stress trials in field proved that the capability of drought-resistance of the transgenic tobaccos were improved. All this have made a firm foundation for the gene engineering research of drought-resistance in plant and will be useful in producing trehalose by plant bioreactors.