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Differential expression analysis of genes involved in Bixin biosynthesis in Bixa orellana L.

Akshatha , Venugopalan (2011) Differential expression analysis of genes involved in Bixin biosynthesis in Bixa orellana L. PhD thesis, University of Mysore.

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Abstract

Bixa orellana L., a tree native to South America, is known for its reddish orange apocarotenoid pigment ‘bixin’ produced only on the aril portion of its seeds. The biosynthetic pathway leading to the synthesis of bixin has been elucidated. It revealed that lycopene is the precursor from which bixin is synthesized through a three step reaction catalyzed by enzymes viz., lycopene cleavage dioxygenase, bixin aldehyde dehydrogenase and norbixin carboxy methyl transferase. Studying the association of these genes with bixin pigment accumulation during Bixa fruit ontogeny and under elicitor mediated stress conditions would be a stepping stone towards understanding regulation of bixin biosynthesis. In addition, it will also facilitate in identifying the key gene(s) involved in bixin biosynthesis. The variation in flowers and fruits of the three B. orellana varieties were identified and the morphological features were documented to assist in the future identification of efficient varieties for large-scale cultivation for annatto dye production. Measurements of fruit characteristics, together with their pigment profile data, were used to identify the superior variety with respect to its pigment yield in the field. Ovate red, conical greenish-red and hemispherical green fruiting varieties were observed. Among those with conical fruit, significant morphological variations were observed and these could be segregated into seven types. Ovate red-coloured fruits were found to be superior in all morphological characteristics, seed yields and total annatto pigment content. To study the bixin gene expression in B. orellana, RNA extraction was standardized using different tissues of Bixa and the same was then used to synthesize cDNA. PCR conditions were standardized for isolation of partial gene fragments of phytoene synthase (psy), lycopene cyclase (lcy-ε), lycopene cleavage dioxygenase (lco), bixin aldehyde dehydrogenase (badh) and norbixin-carboxy methyl transferase (n-cmt). The isolated genes were cloned to T-tail vector for sequencing. The confirmed sequences of psy, lco, badh, n-cmt and lcy-ε were submitted to NCBI. Annatto pigment was analysed in different tissues of three Bixa varieties and it was found to be significantly different in all three varieties. Seeds of ovate variety recorded the highest annatto pigment content while it was lowest in the hemispherical variety. Expression of lco, badh, n-cmt, psy and lcy-ε genes were in accordance with the annatto pigment profile of different tissues of three Bixa varieties. Analysis of annatto pigment and bixin gene expression during ontogeny of ovate variety of Bixa fruit clearly showed that there is a positive relation between gene expression and pigment accumulation. In order to study bixin gene expression in a more controlled environment, an alternate mode was developed for annatto pigment production. Normal root cultures capable of producing 0.346% of annatto pigment in vitro were established. Annatto pigment content in these normal root cultures were augmented using both biotic and abiotic elicitors. Aqueous extract of Rhizopus oligosporus and Methyl jasmonate enhanced the annatto pigment production in root cultures by 1.2 folds. Studying bixin gene expression under elicitor induced stress conditions in both standing Bixa crop and normal root cultures of Bixa showed that bixin gene expressions were up-regulated thereby augmenting annatto pigment content. Based on the studies carried out during fruit development in normal and elicited conditions and under controlled environment using normal root cultures, Lycopene cleavage dioxygenase was identified as the key gene involved in bixin biosynthesis in B. orellana. In addition to identifying the key gene in bixin biosynthesis, the antisense construct of partial gene fragments of lco, badh and n-cmt were prepared and cloned into pCAMBIA 1304 binary vector. The recombinant binary vectors were then transformed to Agrobacterium tumefaciens. Agrobacterium mediated in vitro transformation of Bixa somatic embryos using pCAMBIA 1304 vector harboring bixin biosynthetic genes showed their transient expression.

Item Type: Thesis (PhD)
Uncontrolled Keywords: Bixa orellana L, reddish orange apocarotenoid pigment, bixin biosynthesis, Annatto pigment
Subjects: 600 Technology > 05 Chemical engineering > 01 Biotechnology and Bioengineering
600 Technology > 08 Food technology > 14 Physical properties > 01 Colours
Divisions: Plant Cell Biotechnology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 13 Nov 2013 04:23
Last Modified: 13 Nov 2013 04:23
URI: http://ir.cftri.com/id/eprint/11279

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