Function Prediction And Transgenic Tobacco Identification Of DSG10 Sequence From Dunalliena Salina

Dunalliena salina , eukaryotic unicellular green alga, able to proliferate under saline conditions ranging from 0.1 to 5.5 mol/L NaCl. In order to study the salt tolerance mechanism at the expression level, our laboratory made the expression gene chips of Dunalliena salina after high salt shock. A series of sequences were screened which express differently under distinct salt conditions.In this thesis, the function of one sequence DSG10 was predicted by bioinformatics analysis, the transgenic tobacco of DSG 10 was obtained:1.DSG10 was analyzed by bioinformatics methods, including open reading frame (ORF) analysis, BLAST, protein secondary structure prediction and phylogenetic analysis. The sequence probably code a nitrate transporter in the membrane.2.Firstly, the vector pCAMBIA2301G with two gus reporter genes was constructed. Then one gus was substituted with the coding sequence of DSG 10 obtained by PCR, and pCAMBIA2301G-DSG10 was constructed. Finally the pCAMBLA2301G-DSG10 was introduced into Agrobacterium tumefaciens using the freeze-thaw method3. After infected by A. tumefaciens, the leaf discs of tobacco were selected on plates containing MS inducement medium supplemented with 75mg/L kanamycin. Regenerated plants were transplanted into vermiculite: sand (1:1) medium.4.The regenerated plants were identified by PCR and Southern blot analysis. PCR analysis showed that approximate 9% tested plants produced the target band. A few of the 9% plants produced the band in Southern blot analysis. Additionally, the number of inserted copies of DSG 10 was different in different transgenic plants.