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Computational analysis of common bean (Phaseolus vulgaris L., genotype BAT93) lycopene ?-cyclase and ?-carotene hydroxylase gene's cDNA.

ABSTRACT: The identification of genes and understanding of genes' expression and regulation in common bean (Phaseolus vulgaris L.) is necessary in order to strategize its improvement using genetic engineering techniques. Generation of expressed sequence tags (ESTs) is useful in rapid isolation, identification and characterization of the genes. To study the gene expression in P. vulgaris pods tissue, ESTs generation work was initiated. Early stage and late stage bean-pod-tissues cDNA libraries were constructed using CloneMiner cDNA library construction kit. In total, 5972 EST clones were isolated using random method of gene isolation. While processing ESTs, we found lycopene ?-cyclase (PvLCY-?) and ?-carotene hydroxylase (PvCHY-?) gene's cDNA. In carotenoid biosynthesis pathway, PvLCY-? catalyzes the production of carotene; and PvCHY-? is known to function as a catalyst in the production of lutein and zeaxanthin. To understand more about PvLCY-? and PvCHY-?, both strands of both cDNA clones were sequenced using M13 forward and reverse primers. Nucleotide and deduced protein sequences were analyzed and annotated using online bioinformatics tools. Results showed that PvLCY-? and PvCHY-? cDNAs are 1639 and 1107 bp in length, respectively. Analysis results showed that PvLCY-? and PvCHY-? gene's cDNA contains an open reading frame (ORF) that encodes for 502 and 305 amino acid residues, respectively. The deduced protein sequence analysis results also showed the presence of conserved domains needed for PvLCY-? and PvCHY-? functions. The phylogenetic analysis of both PvLCY-? and PvCHY-? proteins showed it's closeness with the LCY-? and CHY-? proteins from Glycine max, respectively. The nucleotide sequence of PvLCY-? and PvCHY-? gene's cDNA and it's annotation is reported in this paper.

SUBMITTER: Bhore SJ

PROVIDER: S-EPMC3602890 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Computational analysis of common bean (Phaseolus vulgaris L., genotype BAT93) lycopene β-cyclase and β-carotene hydroxylase gene's cDNA.

Bhore Subhash Janardhan SJ Amelia Kassim K Wang Edina E Priyadharsini Sindhuja S Shah Farida Habib FH

Bioinformation 20130221 4

The identification of genes and understanding of genes' expression and regulation in common bean (Phaseolus vulgaris L.) is necessary in order to strategize its improvement using genetic engineering techniques. Generation of expressed sequence tags (ESTs) is useful in rapid isolation, identification and characterization of the genes. To study the gene expression in P. vulgaris pods tissue, ESTs generation work was initiated. Early stage and late stage bean-pod-tissues cDNA libraries were constru ...[more]

PMID: 23519320

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Project description:BACKGROUND:Common bean (Phaseolus vulgaris L.) is an important part of the human diet and serves as a source of natural products. Identification and understanding of genes in P. vulgaris is important for its improvement. Characterization of expressed sequence tags (ESTs) is one of the approaches in understanding the expressed genes. For the understanding of genes expression in P. vulgaris pod-tissue, research work of ESTs generation was initiated by constructing cDNA libraries using 5-day and 20-day old bean-pod-tissues. Altogether, 5972 cDNA clones were isolated to have ESTs. While processing ESTs, we found a transcript for calmodulin (CaM) gene. It is an important gene that encodes for a calcium-binding protein and known to express in all eukaryotic cells. Hence, this study was undertaken to analyse and annotate it. OBJECTIVE:The objective of this study was to analyze and annotate P. vulgaris CaM (PvCaM) gene cDNA and its deduced protein (amino acids) sequence. MATERIALS AND METHODS:Both strands of PvCaM cDNA clone were sequenced using M13 forward and reverse primer to elucidate the nucleotide sequence. The cDNA sequence and deduced protein sequence were analyzed and annotated using bioinformatics tools available online. The secondary structures and three-dimensional (3D) structure of PvCaM protein were predicted using the Phyre automatic fold recognition server. RESULTS:Results showed that PvCaM cDNA is 818 bp in length. The cDNA analysis results showed that it contains an open reading frame that encodes for 149 amino acid residues. The deduced protein sequence analysis results showed the presence of conserved domains required for CaM function. The predicted secondary structures and 3D structure are analogous to the Solanum tuberosum CaM. CONCLUSIONS:This study analyzed and annotated PvCaM cDNA and protein. However, in order to obtain a complete understanding of PvCaM protein, further study on its expression, structure and regulation is essential.

Project description:Traditional yellow maize though contains high kernel carotenoids, the concentration of provitamin A (proA) is quite low (<2 ?g/g), compared to recommended level (15 ?g/g). It also possesses poor endosperm protein quality due to low concentration of lysine and tryptophan. Natural variant of crtRB1 (?-carotene hydroxylase) and lcyE (lycopene-?-cyclase) cause significant enhancement of proA concentration, while recessive allele, opaque2 (o2) enhances the level of these amino acids. Development of biofortified maize enriched in proA, lysine and tryptophan thus holds significance in alleviation of micronutrient malnutrition. In the present study, marker-assisted stacking of crtRB1, lcyE and o2 was undertaken in the genetic background of four maize hybrids (HQPM1, HQPM4, HQPM5, and HQPM7) popularly grown in India. HP704-22 and HP704-23 were used as donors, while four elite QPM parents viz., HKI161, HKI163, HKI193-1, and HKI193-2 were used as recipients. CrtRB1 showed severe segregation distortion, while lcyE segregated as per the expectation. Recovery of recurrent parent genome (RPG) among selected backcross progenies ranged from 89 to 93%. Introgressed progenies possessed high concentration of proA (7.38-13.59 ?g/g), compared to 1.65-2.04 ?g/g in the recurrent parents. The reconstituted hybrids showed an average of 4.5-fold increase in proA with a range of 9.25-12.88 ?g/g, compared to original hybrids (2.14-2.48 ?g/g). Similar plant-, ear-, and grain- characteristics of improved versions of both inbreds and hybrids were observed when evaluated with their respective original versions. Mean lysine (0.334%) and tryptophan (0.080%) of the improved hybrids were at par with the original versions (lysine: 0.340%, tryptophan: 0.083%). Improved hybrids also possessed similar grain yield potential (6,301-8,545 kg/ha) with their original versions (6,135-8,479 kg/ha) evaluated at two locations. This is the first study of staking crtRB1-, lcyE-, and o2-, favorable alleles in single genetic background. The improved inbreds can be effectively used as potential donor for independent and/or simultaneous introgression of crtRB1, lcyE, and o2 in the future breeding programme. These biofortified maize hybrids, rich in proA, lysine and tryptophan will hold great promise for nutritional security.

Dataset Information

Computational analysis of common bean (Phaseolus vulgaris L., genotype BAT93) lycopene ?-cyclase and ?-carotene hydroxylase gene's cDNA.

Publications

Computational analysis of common bean (Phaseolus vulgaris L., genotype BAT93) lycopene β-cyclase and β-carotene hydroxylase gene's cDNA.

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