Identifying a Carotenoid Cleavage Dioxygenase 4a Gene and Its Efficient Agrobacterium-Mediated Genetic Transformation in Bixa orellana L

• Published in: Applied biochemistry and biotechnology 2016-07, Vol.179 (5), p.697-714
• Main Authors: Sankari, Mohan, Hemachandran, Hridya, Anantharaman, Amirtha, Babu, Subramanian, Madrid, Renata Rivera, C, George Priya Doss, Fulzele, Devanand P., Siva, Ramamoorthy
• Format: Article
• Language: English
• Subjects: Agrobacterium - genetics; Agrobacterium tumefaciens; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Bacteria; Biochemistry; Biosynthesis; Biotechnology; Bixa orellana; Bixaceae - enzymology; Bixaceae - genetics; Carotenoids; Carotenoids - genetics; Carotenoids - metabolism; Chemistry; Chemistry and Materials Science; Cosmetics; Crocus; Crocus - enzymology; Crocus - genetics; Dioxygenases - genetics; Dioxygenases - metabolism; Economic importance; Enzymes; Food dyes; Genetic engineering; Oxygenases - genetics; Oxygenases - metabolism; Phylogenetics; Phylogeny; Pollination; Transformation, Genetic
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-016-2025-8

Carotenoids are metabolized to apocarotenoids through the pathway catalysed by carotenoid cleavage oxygenases ( CCOs ). The apocarotenoids are economically important as it is known to have therapeutic as well as industrial applications. For instance, bixin from Bixa orellana and crocin from Crocus sativus are commercially used as a food colourant and cosmetics since prehistoric time. In our present study, CCD4a gene has been identified and isolated from leaves of B. orellana for the first time and named as Bo CCD4a; phylogenetic analysis was carried out using CLUSTAL W. From sequence analysis, Bo CCD4a contains two exons and one intron, which was compared with the selected At CCD4, Rd CCD4, Gm CCD4 and Cm CCD4a gene. Further, the Bo CCD4a gene was cloned into pCAMBIA 1301, transformed into Agrobacterium tumefaciens EHA105 strain and subsequently transferred into hypocotyledons and callus of B. orellana by agro-infection. Selection of stable transformation was screened on the basis of PCR detection by using GUS and hptII specific primer, which was followed by histochemical characterization. The percent transient GUS expression in hypocotyledons and callus was 84.4 and 80 %, respectively. The expression of Bo CCD4a gene in B. orellana was confirmed through RT-PCR analysis. From our results, the sequence analysis of Bo CCD4a gene of B. orellana was closely related to the Cs CCD4 gene of C. sativus , which suggests this gene may have a role in various processes such as fragrance, insect attractant and pollination.