Tibetan Rhodiola Check The Chalcone Synthase Gene Cloning And Analysis

The flavonoids of Rhodiola fastigiata had many pharmaceutical properties for human health. The studies on increasing flavonoids in plants had been more and more popular and important. Until now, many methods had been carried out on increasing flavonoids in plants, and it was a available method via physiological and biochemical to regulate flavonoids content. At the same time, it will be an available method by gene engineering to increase flavonoids content in the future. There were many enzymes in flavonoids metablic synthesis pathway, but chalcone synthase (CHS) was the key enzyme. So, carring out the studies on these two enzymes was very important valuable for theory and application.DNA and RNA were isolated respectively from Rhodiola fastigiata (Hook. f. et Thoms.) S. H. Fu by CTAB method, and a CHS full-length cDNA gene fragment was cloned from Rhodiola fastigiata (Hook. f. et Thoms.) S. H. Fu. Temporal expression of CHS gene was analysed. In order to study the interrelation of expression level of CHS gene, a detailed investigaton and analysis was carried out. The results as following:1. The DNA and RNA were isolated respectively from Rhodiola fastigiata (Hook. f. et Thoms.) S. H. Fu by CTAB method. The result showed that DNA was high quality. RNA obtained from Rhodiola fastigiata (Hook. f. et Thoms.) S. H. Fu, which A260/A280 approximated to 2.0, these results indicated that RNA was high purity and good quality. The CTAB method was especially useful for the DNA and RNA extraction of plant materials that contained plenty of polysaccharide and polyphenol.2. The genomic DNA sequence of CHS gene fragment was cloned from Rhodiola fastigiata (Hook. f. et Thoms.) S. H. Fu by using a pair degenerate primers. Which was 860 bp long, and GenBank accession number was EF070214.3. A new complete DNA sequence of the chalcone synthase (CHS) gene, which is specially expressed in Rhodiola sachalinensis, was cloned by method of single oligonucleotide nested PCR(SON-PCR). The gene, designated Rhchs, contained a length of 2 443 bp including a coding region of 381 amino acids. CHS protein predicted from Rhchs showed 87 %, 87 %, 86 %, 85 % identities with CHS of Vitnus vinifera , Camellia sinensis , Rhododendron simsii, Petunia×hybrida, and retained most of the conserved residues. Some putative cis elements characteristic of the CHS gene were found at the promoter region of Rhchs.4. Phylogenetic analysis of Rhchs coding genes in general revealed that Rhchs has high similarity with the other 10 plants.5. Structure and organization of Rhchs analysis show that the Rhchs genes were comprised of 2 extrons and 1 intron. The ATG start codon and the TAA stop codon located on the first extron and the second extron respectively. The second extron was larger but less variation than the first intron.6. According to the sequence of Rhchs, the expression vector pZP211: Rhchs was constructed.7. The content of the total flavonoid in Rhodiola fastigiata (Hook. f. et Thoms.) S. H. Fu was experimented, and the result showed that we can get 4.6 mg/g in every Rhodiola fastigiata (Hook. f. et Thoms.) S. H. Fu.