Cloning Of Trehalose-6-phosphate Synthase Gene (TPS1) And Transformation Of Strawberry With TPS1

Drought is the major adverse environmental factor that influences on plant growth and development, and decreases crops productivity significantly. With the development of molecular biology, people find another way of cultivar new breeds.Trehalose, a non-deoxidize disaccharide, serves as a protectant of enzymes and membranes in many microorganism, algae and lower invertebrate animals against various stress. In yeast, the synthesis of trehalose is catalyzed by trehalose -6-phosphate synthase/phosphatase complex, which is encoded by four genes: TPSl, TPS2, TPS3 and TSL1. TPS1 is a functional trehalose-6-phosphate synthase that converts UDP-glucose and glucose-6-phosphate to trehalose- 6- phosphate, and TPS2 encodes the phosphatase, which dephosphorylates trehalose -6-phosphate, and TSL1 together with TPS3 is supposed to stabilize and regulate the complex. Experiment shows that TPSl gene can endow organism the ability of synthesis trehalose, the dephosphorylation of the trehalose-6-phosphate is not special, and it can be replaced by other phosphatases.The TPSl gene from Saccharomyces cerevisiae was cloned by PCR amplification. The 1.488kb DNA fragment was sequenced and ligated to pBI121 vector and transformed into otsA deficient of E.coli strains (FF4169). OtsA gene is in charge of trehalose-6-phosphate synthesis in E.coli.In growth curve experiment, the transformants that carried the 1.488kb DNA fragment grew well in minimum medium, which contains 0.5mol/L NaCl, while control strains couldn't endure it. From the result, we could conclude that when transformed into E.coli strains defective in otsA gene, the TPSl gene of S.cerevisiae was able to restore otsA gene's functions during salt stress.Based on the work above, TPSl gene was transformed into Agrobacterium tumefaciens (strain LBA4404), and then introduced into strawberry. After selecting kanamycin resistant transformants on MS medium, their DNA were extracted for PCR and dot blot. In PCR, the 1.488kb fragment was amplified, and the result of dot blot was positive.