Molecular Cloning And Prokaryotic Expression Of Chalcone Synthase Gene In Fressia Hybrida

Fressia hybrida belongs to freesia genus of Iridaceae which is a kind of perennial herbaceous plant. Fressia hybrida is a popular cut-flower for the beauty and strong fragrance of the flowers. Chalcone synthase (CHS) catalyzes the first committed step in the biosynthesis of flavonoids, it plays an important role in pigment production. In this research, the cDNA and DNA sequence of chalcone synthase gene were cloned from Fressia hybrida. This provides useful information for further study in flower color of plant.The main results were as follows:1. A total of 165 recombinant clones were randomly chosen from the cDNA library of red Fressia hybrida petal. 137 high-quality expressed sequence tags were obtained by single-pass sequencing, and the sequences size ranged from 370bp to 730bp. 85 unique sequences were formed by cluster analysis. By the blastx and fastx analysis, 79 unique sequences(93%) exhibited homology with the non-redundant protein sequences database. 4 transcripts appeared to encode enzymes that related with pigment production, one transcript was chalcone synthase gene 3'sequence. Homology searches showed that 6 unique sequences have no similarity to any other protein sequences in public databases. These genes might have specific roles in Fressia hybrida.2. Base on the method of EST sequence analysis and RACE, the cDNA sequence of chalcone synthase gene was isolated from petals of red Fressia hybrida. This sequence was named FhCHS, 1240bp in length, its Genbank accession number was JF732897. The complete open reading frame was 1170 bp and encoded 389 amino acids. The putative FhCHS with a calculated molecular weight of 42.6 kDa and an isoelectric point of 5.74. Homology analysis and molecular phylogenetic analysis between putative FhCHS and other plants CHS showed that FhCHS is a member of the large family of plant CHSs.3. Using genomic DNA as template, the DNA sequence of chalcone synthase gene was cloned from Fressia hybrida. This sequence was 1369bp long and composed of two exons and one intron, one of the typical features of chalcone synthase genes. The intron, 129bp long, which was inserted in the sixty cysteine codon. The intron was also found that the putative splicing site obeyed the GT/AG rule. Genbank accession number of the DNA sequence was JF732898.4. The recombinant FhCHS protein was successfully expressed in Escherichia coli strain BL21(DE3) with pET-28a(+) vector and the result showed that molecular weight of the expressed FhCHS protein had about 44 kDa, a size matching that of the predicted by bioinformatic analysis. The amount of gene expression reached the maximum 4.5h after induction, and the inducing concentration of IPTG was 0.4mM.