Trehalose-6-phophate Synthase Gene Plant Expressing Vector's Construction And Transformation

Based on cDNA of tps1 gene, pcAMBIA1303 plasmid, we have succeeded to construct plant reombinant vector PHFSM001Transforming the construct plant expressing vector to immature of wheat embryo by gene gun, the wheat plant possessing the tpsl gene is attained by tissue culture and hygromylin selected.Using the tpsl gene digested form the prokaryotic expressing vector as template , according to the published sequence, PCR primers of tpsl gene was designed , 1500bp fragment of tpsl was generated . Recovered the agarose and identified by agarose gelelectrophoresis .The producys of PCR fragments and pcambial303 plasmid Ligation were transformed into E. coli(DH5a). The result of PCRof positive recombinant and restriction analysis demonstrated that the plant expressing vector of tps is attained.The tpsl gene was introguced into immature embryo cells of 12-16 days Wheat cultivars wen . 6 via gene gun delivery technique . Resistantcalli were selected on medium containing hygromin (40-60mg/l). Four green plants were regenerated form resistant calli of wen. 6 derived form 1500 implanted embryos .No green plant was regenerated form calli of 200 non-transformed embryos . PCR assays of 4 green plants showed that two of them attained the expected size of amplified DNA fragment (1500bp). Southern blot of 4 green plants confirmed stable integration of tpsl gene into wheat genome.