Molecular structure and the second introns variation of gene F3'H of two medicinal Chrysanthemum morifolium populations

The gene F3'H encodes one of the key enzymes controlling the biosynthesis of flavonoids in higher plants. In this study, two methods-long fragment cloning and homology cloning-were used to obtain full-length cDNAs and partial genomic DNA sequences for the F3'H genes in two medicinal chrysanthemum populations, 'Chu-ju' and 'Gong-ju'. Analysis of gene structure showed that in both populations the F3'H gene contained three exons and two introns, with the 5′ and 3′ ends of the introns conforming to the GT/AG rule. The F3'H genes of the two medicinal chrysanthemum populations both encoded 509 amino acids in their open reading frames (ORF) with 98.88% homology, which indicated the gene is highly conserved. Analysis of intron structure showed that the second intron of the F3'H genes in the two populations contained motifs closely related to gene transcription that were similar to TATA-box, CAAT-box, AT-rich element, MYB binding site, HD-Zip, and HSE motifs. This suggested that introns may be involved in expression regulation of medicinal chrysanthemum F3'H genes. The two medicinal chrysanthemum populations had abundant allelic variation in their intron regions. Besides 23 single-nucleotide polymorphisms (SNPs), 'Gong-ju' had a 39 bp insertion sequence compared with 'Chu-ju', and there were four or six structures similar to the TATA-box and one CAAT-box in this sequence. In addition, in another site, 'Gong-ju' had two G-box structures related to light response regulation that 'Chu-ju' did not have. These intron variations may be related to physiological differences between the two populations; in 'Gong-ju' the expression of F3'H is higher, the yellow tubular florets are smaller, the content of flavonoids like apigenin is higher, and the content of luteolin is lower than in 'Chu-ju'. Therefore, it can be speculated that the intron sequence of the F3'H gene in medicinal chrysanthemum is a positive control element for expression of the gene.