Fused gene of Streptococcus mutans'PacP and CtxB transformation to tomato

Objective: To obtain the transgenic tomato plants with fused gene of pacP and ctxB. Methods: Sterilized tomato seeds with 3-4% sodium hypochlorite solution. Germinated the seeds on TMT1 medium. The tomato cotyledons of 6-8 days were pre-cultured in feeder layer. Agrobacterium tumefaciens clonie was grown in LB liquid medium supplemented with 20mg/L rifamycin and 100mg/L kanamycin, and incubated overnight at 28℃ with shaking 200rpm. Take 20µl overnight Agrobacterium tumefaciens in 20ml YM selective medium supplemented with 20µl acetosyringone, incubated at 28℃ with shaking untill OD600 got to 0.6-0.7. Centrifuged at 8000rpm for 10min at 20℃.Discarded the supernatant and resuspended the pellet in TMT2 liquid medium by vortexing. Inoculum was ready to be used. The explants were immersed in Agrobacterium tumefaciens for 30min, after that co-cutured in KCMS solid medium. Put the explants on selective differentiated media with kanamycin (100mg/L). Regeneration of the tomato buds resistant to Km. According to the sequence of gusA and nptⅡgenes, the primers were designed for amplifying the two gene fragments. Detection of the resistant plants via GUS Histochemical Assay and PCR test of fused gene and fragment of fused gene. Results:The gusA segment, nptⅡsegment and the fused genes of the tomato transformants were found by PCR, and the detection via GUS Histochemical Assay appeared opsitive.All results indicated that the transgenic tomato plants were obtained and the fused gene were integrated in the tomato genome successfully. Conclusions:The fused gene were integrated in the tomato genome successfully, and obtained the transgenic tomato plants successfully.