Molecular Cloning Chalcone Synthase Gene And Studies On Genetic Transformation Of Quamocliy Pennata By CHS Gene

Chalcone Synthase (CHS) is a key enzyme in the biosynthesis of all classes of flavonoids. Increase or decrease its expression in plant would change flower color. We cloned the cDNA sequence of CHS gene from Narcissus by RT-PCR and constructed plant expression vector p1301BC by one median clone. At the same time, we transfered CHS to Quamocliy pennata by Agrobacterium mediated and established genetic transformation system of Quamocliy pennata through the guide of GUS reporter gene. The main results are as below.1. We extracted RNA from Narcissus, cloned the cDNA sequence of CHS gene by RT-PCR and analyzed the coding sequence of gene. The result demonstrates that the sequence of the coding region is 1167bp,encodes a protein of 389 amino acid. The homology between Quamocliy pennata CHS's cDNA and other plant's is above 80%, 85% between amino acid.2. In order to express CHS in plant, we constructed plant expression vector p1301B Q C. First added Camv 35s promoter and nos terminator to CHS by one median clone. And then ligated the whole fragment into plant expression vectorpCAMBIA-1301. The result of identification of restriction Sites of p1301B Q C and the sequence examination showed that the construction was successful.3. Tissue culture procedure in the Quamocliy pennata genetic transformation was developed. Cotyledon produce callus in MS+6-BA 0.5 mg/L+254-D 2 mg/L and hypocotyl produce it in MS+6-BA 1 mg/L+NAA 0.5 mg/L. Callus initiate in MS+6-BA 2 mg/L.4. Established efficient transgenic technology of Quamocliy pennata. Gus examinaton showed that the rate of transgene was higher. And PCR analysis ,PCR-Southem and Southern analysis all showed that CHS was intigrated into Quamocliy pennata chromosome successfully.