Identification and sequencing of CCD4a and CCD4b genes in wild saffron

Apocarotenoid compounds cause various communication functions in plants. Apocarotenoids are the result of enzymatic cleavage of carotenoids catalyzed by carotenoid dioxygenases (CCD). The CCD4 family is the largest family of plant CCDs. In this research, genomic DNA was extracted from fresh leaves of wild saffron Crocus haussknechtii BOISS and used as a template for amplification of CCD4a and CCD4b genes in PCR reaction. PCR products were sequenced after purification. Then, the gene sequence was compared with the saffron gene available in the gene bank and a phylogenetic tree related to their sequence was drawn. In addition, the amino acids of proteins were compared and the spatial structure of the protein of these two genes was drawn. The results showed that the number of nucleotides in CCD4a gene is equal to 2402 and in CCD4b gene is 2373 kb. The dendrogram related to the sequence of CCD4a gene showed that the species were divided into 5 separate groups based on the similarity in the sequence of nucleic acids, and the highest similarity was related to the CCD4a gene of Iranian agricultural saffron with a rate of 99.82. In addition, the dendrogram diagram for the CCD4b gene was divided into three groups, with the lowest distance between this gene and the genes of saffron species. By defining the exon regions, the protein sequence of CCD4a and CCD4b genes was determined, and their number of amino acids was equal to 577 and 567, respectively. The findings of this research can provide valuable information regarding the behavior and reaction of CCD4a and CCD4b enzymes in the synthesis of C. haussknechtii apocarotenoids and can be used in gene transfer programs from wild saffron to saffron be a useful crop.