The Beet Sucrose Phosphate Synthase (sps) Gene And Its Genetic Transformation

Sugar Beet is one of our country sugar crops,mainly grow in north China, in the breeding process, the main purpose is to cultivate high yield,high sugar,high quality varieties sugar beet. In the production of sugar beet there are more diseases and insect pests, thus affecting the quality of the sugar beet. In order to change the plant growth character, genetic engineering is the most efficient means. Because genetic engineering has the advantages of cycle short,quick results. So genetic engineering is an effective way in new varieties of plants.Due to establish sugar beet plant regeneration system more difficult, genetic transformation efficiency is low, repeatability is bad, therefore, sugar beet plant regeneration and genetic transformation system is established, produced high quality, high yield of sugar beet vareties is the goal of many researchers, with a great significance.This research is through the RT-PCR, sugar beet SPS gene has been cloned, constructed restructured plasmid, Using Agrobateium-mediated transformation is going to purpose gene to lead sugar beet, converting conditions are optimized, this study established stable transformation system, get of SPS gene turned sugar beet of regenerative plants.The main results as follows:l.SPS gene amplificationUse of RT-PCR from sugar beet leaves piece of RNA cloned beet SPS gene. Sequence determination, the gene has100%homology with SPS gene nucleic acid sequence.2. The construction of the plant expression vectorUse pMD18-T Vector cloning vector, SPS gene and plasmid pBI121were connected, this gene was controlled by CaMV35S promoter and Tnos terminator.the vector with selective marker npt II.3.Optimized of Agrobacterium-mediated transformation for sugar beet petioles and genetic transformation systemThe results shows:Growth to growing period of logarithm A.tumefaciens diluted, determine the concentration of OD6000.6; After48h of the petiole training, and5min for infection; lOOμmol/L AS in co-cultivation;22℃~25℃trained4days; After cefazolin with antibacterial,200mg/L Kanamycin concentration of screening.4. Test positive for plantSugar beet SPS gene trough Agrobaterium-mediated transformation.27strains of Kan resistance for sugar beet stable regeneration plant, extraction and plant genomic DNA, the PCR detection has8strains positive plants, PCR positive rate was29%.