Transformation Of Tobacco By Using Serine Racemase Of Maize As A Novel Selection Marker

Serine racemase is a bifunctional enzyme which catalyzes not only serine （L-serine and D-serine） racemization but also dehydration of L-serine and D-serine to private. In this study, serine racemase gene from Zea mays by Agrobacterium-mediated tobacco leaves and callus genetic transformation, while GUS gene into tobacco as a control, compared to ZmSR and GUS gene in leaves and callus transforming effect in the conversion of genetic transformation process, laying he foundation for ZmSR as a new and resistance selection marker. The main results are summarized as follow:1、According to searching ZmSR gene prediction sequence （NM₀01143028.1） from NCBI, and that ZmSR gene length of 1303 bp. With Nco I and BstP I digested plasmid verify pCAMBIA 1301-ZmSR, the result is consistent with the expected size, preliminary evidence ZmSR genes connected to the pBI1301 carrier. The plasmid pBI 1301-ZmSR DNA fragments were sequenced consistent with the predicted results of sequencing genes.2、Making the competent cell of A.tumefaciens LBA4404, then transformed the plasmid of pBI1301-ZmSR into A.tumefaciens LBA4404 by liquid nitrogen freeze-thaw method. Digested verify pBI 1301-ZmSR from Agrobacterium, the results are consistent with the expected size. According to forecasts of ZmSR gene sequences, primers were designed to pBI1301-ZmSR original plasmid PCR was performed to verify the results amplified the size of the cord serine racemase gene bands （340bp）, proved that the restructuring plasmids have been successfully introduced into A.tumefaciens can be used for genetic transformation.3、The best suitable medium of tabacco leaves for callus induction was MS+6-BA 0.2 mg/L+2,4-D1.5 mg/L+sucrose 25 g/L+agar 6 g/L; Induced callus induction rate of 100% of the medium, and the fastest, best callus proliferation.4、In this study, a plasmid pBI1301-ZmSR for the purpose gene plasmid pBI121-GUS as a control, D-serine and Kan as a screening reagent for Kan and untransformed plants were resistant to the critical concentration of D-serine screening, respectively. After a critical concentration of antibiotic screening on kanamycin and different concentrations of D-serine medium which that tobacco leaves were kanamycin and neomycin sensitivity test D-serine determined tobacco leaves kanamycin the critical concentration of 50 mg/L, the critical concentration of D-serine is 12 mM. Tobacco callus kanamycin sensitivity test D-serine. To determine the critical concentration of tobacco callus on kanamycin to 30 mg /L, the critical concentration of D-serine is 6 mM.5, Agrobacterium-mediated method pBI1301-ZmSR and pBI121-GUS were transferred into tobacco leaves and callus. It can be get more transformed plants in the conversion process. pBI1301-ZmSR resistance to leaf through molecules detected by screening seedlings have obtained 3 positive plants resistant seedlings obtained from the callus had 6 positive plants.