Cloning and Genetic Transformation of CnCHS Gene from Camellia nitidissima

Abstract

Using total cDNA from the petals of Camellia nitidissima as template,we amplified the full length chalcone synthase( CHS) cDNA of C. nitidissima successfully by the specific primers designed according to the CnCHS sequence in genbank. The amplified fragment of Cn CHS was inserted into PMD18-T vector and then transformed into E. coli DH5α. After the compounded plasmid was identified by enzyme digestion and sequencing,it was digested and connected to the expression vector pCAMBIA1300. The compounded plasmid pCAM-CnCHS was identified and the sense expression vector of CnCHS was constructed successfully. Then,the pCAM-CnCHS was transformed into Agrobacterium tumefaciens EHA105 for plant infections,and 18 transgenic tobaccos containing CnCHS were obtained. The transgenic plants were then identified by PCR and Southern blotting,and the transgenic tobaccos which contain single copy of CnCHS gene were obtained with the positive rate of 67%. The genetic transformation system of CnCHS to tobaccos was constructed successfully,which would lay the foundation for further function research and regulation effects on flower color of CnCHS.