title: Identifying a Carotenoid Cleavage Dioxygenase 4a Gene and Its Efficient Agrobacterium-Mediated Genetic Transformation in Bixa orellana L.
abstract: 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 BoCCD4a; phylogenetic analysis was carried out using CLUSTAL W. From sequence analysis, BoCCD4a contains two exons and one intron, which was compared with the selected AtCCD4, RdCCD4, GmCCD4 and CmCCD4a gene. Further, the BoCCD4a 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 BoCCD4a gene in B. orellana was confirmed through RT-PCR analysis. From our results, the sequence analysis of BoCCD4a gene of B. orellana was closely related to the CsCCD4 gene of C. sativus, which suggests this gene may have a role in various processes such as fragrance, insect attractant and pollination.
authors.affiliation: School of Bio Sciences and Technology, VIT University, Vellore, 632014, Tamil Nadu, India.
authors.forename: Mohan
authors.initials: M
authors.lastname: Sankari
authors.affiliation: School of Bio Sciences and Technology, VIT University, Vellore, 632014, Tamil Nadu, India.
authors.forename: Hridya
authors.initials: H
authors.lastname: Hemachandran
authors.affiliation: School of Bio Sciences and Technology, VIT University, Vellore, 632014, Tamil Nadu, India.
authors.forename: Amirtha
authors.initials: A
authors.lastname: Anantharaman
authors.affiliation: School of Bio Sciences and Technology, VIT University, Vellore, 632014, Tamil Nadu, India.
authors.forename: Subramanian
authors.initials: S
authors.lastname: Babu
authors.affiliation: Cenro de Investigacion Cientifica de Yucatan, A.C. Calle 43 No. 130, Col. Chuburnade Hidalgo, 97200, Merida, Yucatan, Mexico.
authors.forename: Renata Rivera
authors.initials: RR
authors.lastname: Madrid
authors.affiliation: School of Bio Sciences and Technology, VIT University, Vellore, 632014, Tamil Nadu, India.
authors.forename: George Priya Doss
authors.initials: GP
authors.lastname: C
authors.affiliation: Plant Biotechnology and Secondary Metabolites Section, NA&BTD, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India.
authors.forename: Devanand P
authors.initials: DP
authors.lastname: Fulzele
authors.affiliation: School of Bio Sciences and Technology, VIT University, Vellore, 632014, Tamil Nadu, India. rsiva77in@rediffmail.com.
authors.forename: Ramamoorthy
authors.initials: R
authors.lastname: Siva
mesh_terms: D060054:Agrobacterium / Q000235:genetics; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D031212:Bixaceae / Q000201:enzymology / Q000235:genetics; D002338:Carotenoids / Q000235:genetics / Q000378:metabolism*; D027622:Crocus / Q000201:enzymology / Q000235:genetics; D049308:Dioxygenases / Q000235:genetics* / Q000378:metabolism; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D014170:Transformation, Genetic
chemical_list: D029681:Arabidopsis Proteins; D002338:Carotenoids; C004588:bixin; C000592331:CCD4 protein, Arabidopsis; D010105:Oxygenases; C500451:carotenoid oxygenase; D049308:Dioxygenases
keywords: Agro-infection; Apocarotenoids; Bixa orellana; CCD4a; Carotenoid cleavage dioxygenase; RT-PCR