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

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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.

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Abbreviations

CCD:

Carotenoid cleavage dioxygenase

NCED:

9-cis epoxy carotenoid dioxygenase

CCO:

Carotenoid cleavage oxygenase

BLAST:

Basic Local Alignment Sequence Tool

NJ:

Neighbour-joining method

CTAB:

Cetyltrimethyl ammonium bromide

MS:

Murashige and Skoog

LB:

Luria broth

YEP:

Yeast extract peptone

NAA:

α-Naphthaleneacetic acid

2,4-D:

2,4-Dichlorophenoxyacetic acid

BA:

Benzyladenine

35S CaMV:

35S promoter of the cauliflower mosaic virus

GUS:

β-Glucucauronidase

hptII :

Hygromycin phosphotransferase II

OD600:

Optical density at 600 nm

GFP:

Green fluorescence protein

PCR:

Polymerase chain reaction

RT-PCR:

Reverse transcriptase–polymerase chain reaction

X-Gluc:

5-Bromo-4-chloro-3-indolyl-b-d-glucuronic acid

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Acknowledgments

We express our heartfelt gratitude to Science and Engineering Research Board—Department of Science and Technology, New Delhi, India, for the support extended through the project [SR/FT/LS-75/2011]. We express our gratitude to Dr. K. Suthindhiran for the fluorescence microscope. The authors are thankful to the VIT University management for their constant support.

Authors’ Contribution Statement

RS conceived and designed the experiments. MS performed the experiments and wrote the manuscript. GC corrected the bioinformatics part of experiments. HH, DF and AA helped to correct the manuscript. SB and RM reviewed the manuscript. All authors have read and approved the manuscript.

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Authors and Affiliations

  1. School of Bio Sciences and Technology, VIT University, Vellore, 632014, Tamil Nadu, India

    Mohan Sankari, Hridya Hemachandran, Amirtha Anantharaman, Subramanian Babu, George Priya Doss C & Ramamoorthy Siva

  2. Cenro de Investigacion Cientifica de Yucatan, A.C. Calle 43 No. 130, Col. Chuburnade Hidalgo, 97200, Merida, Yucatan, Mexico

    Renata Rivera Madrid

  3. Plant Biotechnology and Secondary Metabolites Section, NA&BTD, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India

    Devanand P. Fulzele

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  1. Mohan Sankari
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Sankari, M., Hemachandran, H., Anantharaman, A. et al. Identifying a Carotenoid Cleavage Dioxygenase 4a Gene and Its Efficient Agrobacterium-Mediated Genetic Transformation in Bixa orellana L.. Appl Biochem Biotechnol 179, 697–714 (2016). https://doi.org/10.1007/s12010-016-2025-8

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