Analysis of beta-carotene hydroxylase gene cDNA isolated from the American oil-palm (Elaeis oleifera) mesocarp tissue cDNA library
Identifieur interne : 000E41 ( Pmc/Corpus ); précédent : 000E40; suivant : 000E42Analysis of beta-carotene hydroxylase gene cDNA isolated from the American oil-palm (Elaeis oleifera) mesocarp tissue cDNA library
Auteurs : Subhash J. Bhore ; Amelia Kassim ; Chye Ying Loh ; Farida H. ShahSource :
- Bioinformation [ 0973-2063 ] ; 2010.
Abstract
It is well known that the nutritional quality of the American oil-palm (
ESTs - expressed sequence tags,
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PubMed: 21364789
PubMed Central: 3040485
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Analysis of beta-carotene hydroxylase gene cDNA isolated from the American oil-palm (Elaeis oleifera) mesocarp tissue cDNA library</title><author><name sortKey="Bhore, Subhash J" sort="Bhore, Subhash J" uniqKey="Bhore S" first="Subhash J" last="Bhore">Subhash J. Bhore</name><affiliation><nlm:aff id="A1">1Molecular Biology Division, Melaka Institute of Biotechnology, Lot 7, Melaka International Trade Centre City, 75450 Ayer Keroh, Melaka, Malaysia;</nlm:aff></affiliation><affiliation><nlm:aff id="A2">Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong-Semeling Road, Bedong, 08100, Kedah Darul Aman, Malaysia;</nlm:aff></affiliation></author><author><name sortKey="Kassim, Amelia" sort="Kassim, Amelia" uniqKey="Kassim A" first="Amelia" last="Kassim">Amelia Kassim</name><affiliation><nlm:aff id="A1">1Molecular Biology Division, Melaka Institute of Biotechnology, Lot 7, Melaka International Trade Centre City, 75450 Ayer Keroh, Melaka, Malaysia;</nlm:aff></affiliation></author><author><name sortKey="Loh, Chye Ying" sort="Loh, Chye Ying" uniqKey="Loh C" first="Chye Ying" last="Loh">Chye Ying Loh</name><affiliation><nlm:aff id="A2">Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong-Semeling Road, Bedong, 08100, Kedah Darul Aman, Malaysia;</nlm:aff></affiliation></author><author><name sortKey="Shah, Farida H" sort="Shah, Farida H" uniqKey="Shah F" first="Farida H" last="Shah">Farida H. Shah</name><affiliation><nlm:aff id="A1">1Molecular Biology Division, Melaka Institute of Biotechnology, Lot 7, Melaka International Trade Centre City, 75450 Ayer Keroh, Melaka, Malaysia;</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">21364789</idno><idno type="pmc">3040485</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3040485</idno><idno type="RBID">PMC:3040485</idno><date when="2010">2010</date><idno type="wicri:Area/Pmc/Corpus">000E41</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Analysis of beta-carotene hydroxylase gene cDNA isolated from the American oil-palm (Elaeis oleifera) mesocarp tissue cDNA library</title><author><name sortKey="Bhore, Subhash J" sort="Bhore, Subhash J" uniqKey="Bhore S" first="Subhash J" last="Bhore">Subhash J. Bhore</name><affiliation><nlm:aff id="A1">1Molecular Biology Division, Melaka Institute of Biotechnology, Lot 7, Melaka International Trade Centre City, 75450 Ayer Keroh, Melaka, Malaysia;</nlm:aff></affiliation><affiliation><nlm:aff id="A2">Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong-Semeling Road, Bedong, 08100, Kedah Darul Aman, Malaysia;</nlm:aff></affiliation></author><author><name sortKey="Kassim, Amelia" sort="Kassim, Amelia" uniqKey="Kassim A" first="Amelia" last="Kassim">Amelia Kassim</name><affiliation><nlm:aff id="A1">1Molecular Biology Division, Melaka Institute of Biotechnology, Lot 7, Melaka International Trade Centre City, 75450 Ayer Keroh, Melaka, Malaysia;</nlm:aff></affiliation></author><author><name sortKey="Loh, Chye Ying" sort="Loh, Chye Ying" uniqKey="Loh C" first="Chye Ying" last="Loh">Chye Ying Loh</name><affiliation><nlm:aff id="A2">Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong-Semeling Road, Bedong, 08100, Kedah Darul Aman, Malaysia;</nlm:aff></affiliation></author><author><name sortKey="Shah, Farida H" sort="Shah, Farida H" uniqKey="Shah F" first="Farida H" last="Shah">Farida H. Shah</name><affiliation><nlm:aff id="A1">1Molecular Biology Division, Melaka Institute of Biotechnology, Lot 7, Melaka International Trade Centre City, 75450 Ayer Keroh, Melaka, Malaysia;</nlm:aff></affiliation></author></analytic><series><title level="j">Bioinformation</title><idno type="eISSN">0973-2063</idno><imprint><date when="2010">2010</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>It is well known that the nutritional quality of the American oil-palm (<italic>Elaeis oleifera</italic>) mesocarp oil is superior to that of African oil-palm (<italic>Elaeis
guineensis</italic> Jacq. Tenera) mesocarp oil. Therefore, it is of important to identify the genetic features for its superior value. This could be achieved through
the genome sequencing of the oil-palm. However, the genome sequence is not available in the public domain due to commercial secrecy. Hence, we
constructed a cDNA library and generated expressed sequence tags (3,205) from the mesocarp tissue of the American oil-palm. We continued to annotate
each of these cDNAs after submitting to GenBank/DDBJ/EMBL. A rough analysis turned our attention to the beta-carotene hydroxylase (<italic>Chyb</italic>) enzyme
encoding cDNA. Then, we completed the full sequencing of cDNA clone for its both strands using M13 forward and reverse primers. The full nucleotide
and protein sequence was further analyzed and annotated using various Bioinformatics tools. The analysis results showed the presence of fatty acid
hydroxylase superfamily domain in the protein sequence. The multiple sequence alignment of selected <italic>Chyb</italic> amino acid sequences from other plant
species and algal members with <italic>E. oleifera Chyb</italic> using ClustalW and its phylogenetic analysis suggest that <italic>Chyb</italic> from monocotyledonous plant species,
<italic>Lilium hubrid</italic>, <italic>Crocus sativus</italic> and <italic>Zea mays</italic> are the most evolutionary related with <italic>E. oleifera Chyb</italic>. This study reports the annotation of <italic>E. oleifera Chyb</italic>.</p><sec id="ABB"><title>Abbreviations</title><p>ESTs - expressed sequence tags,
<italic>EoChyb</italic> - <italic>Elaeis oleifera beta-carotene hydroxylase</italic>,
MC - main cluster</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Muniran, F" uniqKey="Muniran F">F Muniran</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Asemota, O" uniqKey="Asemota O">O Asemota</name></author><author><name sortKey="Shah, Fh" uniqKey="Shah F">FH Shah</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lange, Bm" uniqKey="Lange B">BM Lange</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ohlrogge, J" uniqKey="Ohlrogge J">J Ohlrogge</name></author><author><name sortKey="Benning, C" uniqKey="Benning C">C Benning</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ronning, Cm" uniqKey="Ronning C">CM Ronning</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sreenivasulu, N" uniqKey="Sreenivasulu N">N Sreenivasulu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Liu, J" uniqKey="Liu J">J Liu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Keith, Cs" uniqKey="Keith C">CS Keith</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Park, Ys" uniqKey="Park Y">YS Park</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hofte, H" uniqKey="Hofte H">H Hofte</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Umeda, M" uniqKey="Umeda M">M Umeda</name></author><author><name sortKey="Uchimiya, H" uniqKey="Uchimiya H">H Uchimiya</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Marchler Bauer, A" uniqKey="Marchler Bauer A">A Marchler-Bauer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Martin, Jf" uniqKey="Martin J">JF Martin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Baroli, I" uniqKey="Baroli I">I Baroli</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vallabhaneni, R" uniqKey="Vallabhaneni R">R Vallabhaneni</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Shah, Fh" uniqKey="Shah F">FH Shah</name></author><author><name sortKey="Cha, Ts" uniqKey="Cha T">TS Cha</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Singh, R" uniqKey="Singh R">R Singh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Arthington, Ba" uniqKey="Arthington B">BA Arthington</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Bioinformation</journal-id><journal-id journal-id-type="publisher-id">Bioinformation</journal-id><journal-title-group><journal-title>Bioinformation</journal-title></journal-title-group><issn pub-type="epub">0973-2063</issn><publisher><publisher-name>Biomedical Informatics</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">21364789</article-id><article-id pub-id-type="pmc">3040485</article-id><article-id pub-id-type="publisher-id">002500052010</article-id><article-categories><subj-group subj-group-type="heading"><subject>Hypothesis</subject></subj-group></article-categories><title-group><article-title>Analysis of beta-carotene hydroxylase gene cDNA isolated from the American oil-palm (Elaeis oleifera) mesocarp tissue cDNA library</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Bhore</surname><given-names>Subhash J</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="aff" rid="A2">2</xref><xref ref-type="corresp" rid="COR1">*</xref></contrib><contrib contrib-type="author"><name><surname>Kassim</surname><given-names>Amelia</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Loh</surname><given-names>Chye Ying</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Shah</surname><given-names>Farida H</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><aff id="A1"><label>1</label>1Molecular Biology Division, Melaka Institute of Biotechnology, Lot 7, Melaka International Trade Centre City, 75450 Ayer Keroh, Melaka, Malaysia;</aff><aff id="A2"><label>2</label>Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong-Semeling Road, Bedong, 08100, Kedah Darul Aman, Malaysia;</aff></contrib-group><author-notes><corresp id="COR1"><label>*</label>Subhash J Bhore: <email>subhashbhore@gmail.com</email>Phone: +60-4-429 8176; Fax: +60-4-429 8109</corresp></author-notes><pub-date pub-type="collection"><year>2010</year></pub-date><pub-date pub-type="epub"><day>20</day><month>9</month><year>2010</year></pub-date><volume>5</volume><issue>3</issue><fpage>104</fpage><lpage>112</lpage><history><date date-type="received"><day>19</day><month>8</month><year>2010</year></date><date date-type="accepted"><day>26</day><month>8</month><year>2010</year></date></history><permissions><copyright-statement>© 2010 Biomedical Informatics</copyright-statement><copyright-year>2010</copyright-year><license license-type="open-access"><license-p>This is an open-access article, which permits unrestricted use, distribution, and reproduction in any medium,
for non-commercial purposes, provided the original author and source are credited.</license-p></license></permissions><abstract><p>It is well known that the nutritional quality of the American oil-palm (<italic>Elaeis oleifera</italic>) mesocarp oil is superior to that of African oil-palm (<italic>Elaeis
guineensis</italic> Jacq. Tenera) mesocarp oil. Therefore, it is of important to identify the genetic features for its superior value. This could be achieved through
the genome sequencing of the oil-palm. However, the genome sequence is not available in the public domain due to commercial secrecy. Hence, we
constructed a cDNA library and generated expressed sequence tags (3,205) from the mesocarp tissue of the American oil-palm. We continued to annotate
each of these cDNAs after submitting to GenBank/DDBJ/EMBL. A rough analysis turned our attention to the beta-carotene hydroxylase (<italic>Chyb</italic>) enzyme
encoding cDNA. Then, we completed the full sequencing of cDNA clone for its both strands using M13 forward and reverse primers. The full nucleotide
and protein sequence was further analyzed and annotated using various Bioinformatics tools. The analysis results showed the presence of fatty acid
hydroxylase superfamily domain in the protein sequence. The multiple sequence alignment of selected <italic>Chyb</italic> amino acid sequences from other plant
species and algal members with <italic>E. oleifera Chyb</italic> using ClustalW and its phylogenetic analysis suggest that <italic>Chyb</italic> from monocotyledonous plant species,
<italic>Lilium hubrid</italic>, <italic>Crocus sativus</italic> and <italic>Zea mays</italic> are the most evolutionary related with <italic>E. oleifera Chyb</italic>. This study reports the annotation of <italic>E. oleifera Chyb</italic>.</p><sec id="ABB"><title>Abbreviations</title><p>ESTs - expressed sequence tags,
<italic>EoChyb</italic> - <italic>Elaeis oleifera beta-carotene hydroxylase</italic>,
MC - main cluster</p></sec></abstract><kwd-group><kwd>African oil-palm</kwd><kwd>American oil-palm</kwd><kwd>fatty acids</kwd><kwd>fatty acid hydroxylase</kwd><kwd>oleic acid</kwd><kwd>sterol desaturase</kwd><kwd>zeaxanthin</kwd></kwd-group></article-meta></front><body><sec id="s1"><title>Background</title><p>Oil‐palm is the major commodity worldwide. The second largest source of
fats and oils to the world market of fats and oils is from <italic>Elaeis guineensis</italic>Jacq Tenera, which is commonly known as African oil-palm. Malaysia is
the main producer and exporter of the palm oil. African oil‐palm is
cultivated on industrial scale due to high palm oil yield derived from its
fruit mesocarp tissue. African oil‐palm, <italic>E. guineensis</italic> Jacq have three
different forms (also called as varieties), namely, ‘Pisifera’, ‘Dura’, and
‘Tenera’. These three forms are distinguished on the basis of shell
thickness of fruits. The commercially cultivated African oil‐palm is a
hybrid from ‘Dura’ (♀) and ‘Pisifera’ (♂)
[<xref ref-type="bibr" rid="R01">1</xref>]. Another oil‐palm species
which is economically less important is <italic>Elaeis oleifera</italic>. This species is also
called as American oil‐palm.</p><p>Palmitic acid, (C16:0) is the predominant fatty acid in palm oil derived from
<italic>E. guineensis</italic> Jacq. Tenera fruit mesocarp tissue. Whereas oil derived from
<italic>E. oleifera</italic> fruit mesocarp tissue is predominant with oleic acid (C18:1)
(68.6%), a fatty acid good for health [<xref ref-type="bibr" rid="R02">2</xref>].
The C16:0 is the major (44%) saturated fatty acid in palm oil derived from
<italic>E. guineensis</italic>. However, the C16:0 content in <italic>E. oleifera</italic>is only 25%. In spite of the high content of the healthy C18:1 in the
<italic>E. oleifera</italic> oil, it is not preferred for commercial plantation due to its poor oil yield.</p><p>The single pass, partial sequencing of randomly isolated anonymous
cDNA clones also called as ESTs has become a rapid and cost-effective
means in gaining information about gene expression and their regulation
[<xref ref-type="bibr" rid="R03">3</xref>]. In addition,
generated ESTs data is useful in new and novel gene’s discovery
[<xref ref-type="bibr" rid="R04">4</xref>],
evaluation of the genome for gene content and its structure,
and for in <italic>silico</italic> comparative expression analysis between different plant
tissues [<xref ref-type="bibr" rid="R05">5</xref>].
Most importantly, ESTs serves as a valuable resource for highthroughput
expression analysis using cDNA-microarray technology [<xref ref-type="bibr" rid="R06">6</xref>]. In
higher plants, numerous genes have been identified by random nucleotide
sequencing of cDNA clones [<xref ref-type="bibr" rid="R07">7</xref>–
<xref ref-type="bibr" rid="R11">11</xref>]. Therefore, in order to study the gene
expression and their patterns in <italic>E. oleifera</italic> fruit mesocarp tissue, ESTs
generation project was initiated. So far, 3,205 ESTs are generated from 17
weeks old <italic>E. oleifera</italic> mesocarp tissue cDNA library (our unpublished
work). Beta carotene hydroxylase (<italic>Chyb</italic>) is one of the isolated cDNA (ESTs) clones.</p><p>By understating potential applications of <italic>Chyb</italic> in genetic engineering of
oil-palm and or in other plants, clone was fully sequenced. The <italic>Chyb</italic> is
involved in zeaxanthin biosynthesis by hydroxylating beta-carotene, but
the enzyme may be involved in other pathways [<xref ref-type="bibr" rid="R12">12</xref>–
<xref ref-type="bibr" rid="R13">13</xref>]. The products of
this enzyme are zeaxanthin and beta-cryptoxanthin
[<xref ref-type="bibr" rid="R14">14</xref>]. Beta-carotene,
zeaxanthin and beta-cryptoxanthin are categorized under carotenoids
which have many industrial applications as food and feed additives, and
are used in cosmetics and as nutraceutical. Currently, vitamin A deficiency
is a global health burden which could be alleviated through provitamin A carotenoid biofortification
in suitable plants [<xref ref-type="bibr" rid="R15">15</xref>]. If we understand in depth
the regulation of carotenoid biosynthesis, enhancement of beta-carotene
could be done by limiting beta-carotene hydroxylation. Therefore in order
to understand more about <italic>EoChyb</italic>, its cDNA clone is analyzed using
computational tools. The nucleotide and protein sequence of <italic>EoChyb</italic>cDNA is analyzed and annotated in this study to find out their features.
The <italic>EoChyb</italic> annotation and features are reported in this paper.</p></sec><sec sec-type="methods" id="s2"><title>Methodology</title><sec id="s2a"><title>cDNA library and EoChyb clone isolation</title><p><italic>Elaeis oleifera</italic>, seventeen week's old [weeks after anthesis (WAA)]
mesocarp tissue cDNA library constructed using the ‘CloneMiner cDNA
library construction kit’ (Invitrogen Corporation) for ESTs generation (our
unpublished work) was used to isolate <italic>EoChyb</italic>. The
<italic>EoChyb</italic> cDNA clone was isolated by random method of cDNA isolation
[<xref ref-type="bibr" rid="R03">3</xref>].</p><p>The cDNA nucleotide sequence reported in this paper has been submitted
to GenBank/DDBJ/EMBL under accession number EU057623.</p></sec><sec id="s2b"><title>Nucleotide sequencing</title><p>The <italic>E. coli</italic> DH5α cells harboring <italic>EoChyb</italic> cDNA were
cultivated in 10 ml LB medium (supplemented with Kanamycin) overnight in dark at 37 °C,
160 rpm. The cultivated <italic>E. coli</italic> DH5α cells were harvested from broth and
plasmid DNA was isolated by using a commercial kit, Wizard® Plus SV
Minipreps DNA purification system (Promega). Sequencing reactions were
carried out for both strands using M13 (Forward); 5’-GTAAAACGACGGCCAG-3’ and M13 (Reverse);
5’-GGATAACAATTTCACACAGG-3’ primers.</p></sec><sec id="s2c"><title>cDNA and protein sequence analysis</title><p>The cDNA sequence was edited manually to eliminate vector and adaptor
sequences from 5’ and 3’ ends. Finalized cDNA sequence analysis was
performed using online free bioinformatics tools. The similarity searches
were performed using blast programs (BlastN, BlastX, and BlastP) against
the databases available at NCBI [<xref ref-type="bibr" rid="R16">16</xref>].
EMBOSS pairwise alignment algorithm [<ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/Tools/emboss/align/">http://www.ebi.ac.uk/Tools/emboss/align/</ext-link>] was used to
compare 2 cDNA sequences to find out homology %. To find out general
features of the cDNA sequence including amino acid composition and
isoelectric point analysis was performed online using bioinformatics tools
available at JustBio [<xref ref-type="bibr" rid="R17">17</xref>].
Guanine and cytosine content (GC %) calculation
was carried out by using ‘DNA/RNA base composition calculator‘, a free
online bioinformatics tool that calculates the molecular mass, elemental
composition, base composition, and percent AT and GC content for
cDNA/DNA/RNA sequences [<xref ref-type="bibr" rid="R18">18</xref>].
Alignment of amino acid sequences and dendrogram construction was carried out using multiple sequence
alignment by ClustalW [<xref ref-type="bibr" rid="R19">19</xref>]
program. Whereas, to find out fully conserved
residues; residues with conserved strong groups and residues with
conserved weak groups in <italic>EoChyb</italic>, amino acid sequences were aligned by
using clustal 2.0.11 multiple sequence alignment program.</p></sec></sec><sec id="s3"><title>Results</title><sec id="s3a"><title><italic>EoChyb</italic> clone isolation</title><p>By random method of cDNA clone isolation, <italic>EoChyb</italic> clone was isolated
from 17 day old mesocarp tissue cDNA library. The serial number of this
randomly isolated cDNA clone in ESTs generation project was 2962;
hence clone identity ‘EoEST-2962’ was given to <italic>EoChyb</italic> cDNA clone.</p></sec><sec id="s3b"><title>Nucleotide sequencing</title><p>The +ve and –ve strand of <italic>EoChyb</italic> cDNA clone were sequenced using
M13 forward and M13 reverse primers, respectively. The <italic>EoChyb</italic> +ve and
–ve strand cDNA sequence after removal of vector and adaptor sequence
was compared and searched for overlaps using blast (bl2seq) program
[<xref ref-type="bibr" rid="R16">16</xref>].
The results produced shows that <italic>EoChyb</italic> cDNA sequence is 1414 bp in
length. By analyzing the finalized cDNA sequence and its deduced amino
acid sequence, <italic>Chyb</italic> identity was given to the cDNA clone.</p></sec><sec id="s3c"><title>cDNA and protein sequence analysis</title><p>The general features of the cDNA nucleotide and protein sequence as
revealed by bioinformatics tools available at JustBio are summarized in
Table 1 (see <xref ref-type="supplementary-material" rid="SD1">supplementary material</xref>).
The annotated nucleotide sequence was submitted to GenBank/DDBJ/EMBL under accession
number EU057623. Whereas the comparison of the <italic>EoChyb</italic> at nucleotide
and protein level with <italic>Chyb</italic> from other plant species and algal members is
depicted in Table 2 (see <xref ref-type="supplementary-material" rid="SD1">supplementary material</xref>).
The nucleotide and deduced amino acid sequences of <italic>EoChyb</italic> cDNA, an open reading frame
(ORF), 5’ and 3’ untranslated regions (UTR), initiation and termination
codon, and the amino acid residues of beta-carotene hydroxylase and sterol
desaturase conserved domains are shown in <xref ref-type="fig" rid="F1">Figure 1</xref>. The <italic>EoChyb</italic>, and
<italic>Chyb</italic> from other selected plants and algal members <italic>Chyb</italic> amino acid
sequence alignment based phylogenetic analysis was carried out and the
constructed dendrogram is shown in <xref ref-type="fig" rid="F2">Figure 2</xref>. Clustal 2.0.11 multiple
sequence alignment program produced results of <italic>EoChyb</italic> amino acid
sequence alignment with amino acid sequences of <italic>Chyb</italic> from other
organisms and showed single fully conserved residues, residues with conserved strong groups and
residues with conserved weak groups (<xref ref-type="fig" rid="F3">Figure 3</xref>).</p></sec></sec><sec id="s4"><title>Discussion</title><p>The availability of <italic>EoChyb</italic> cDNA clone in 17 day old mesocarp tissue
cDNA library indicates that <italic>Chyb</italic> is expressed in developing <italic>E. oleifera</italic>fruit mesocarp tissue. However, the level of its expression, pattern of
expression and tissue specificity is not known. The analysis of developing
fruit mesocarp by Northern blot technique could shade the light on level
and pattern of its expression [<xref ref-type="bibr" rid="R20">20</xref>].
The GC content in <italic>EoChyb</italic> cDNA is
56%. This GC % is close to the predicted GC content in coding sequences
of <italic>Elaeis oleifera</italic> and <italic>Elaeis guineensis</italic>[<xref ref-type="bibr" rid="R21">21</xref>–<xref ref-type="bibr" rid="R22">22</xref>]. Homology analysis using
BlastP indicates that EoChyb protein shows 82-98% homology with <italic>Chyb</italic>from other monocot plant species. Comparison of <italic>EoChyb</italic> with Chyb from
13 dicot plant species shows range of homology from 71-98%. However,
<italic>Chyb</italic> from algal member shows only 78-84% homology with <italic>EoChyb</italic>(Table 2, see <xref ref-type="supplementary-material" rid="SD1">supplementary material</xref>). The relatively low level of
<italic>EoChyb</italic> homology with <italic>Chyb</italic> from algal member is in line with the
evolution in plant species. Monocots are highly evolved in comparison
with the dicots and algae. Conserved domain search in <italic>EoChyb</italic> indicates
that amino acid residue 46-324 are part of the beta-carotene hydroxylase
(fatty acid hydroxylase super family) conserved domain; and amino acid
residues 184-309 belongs to the sterol desaturase [lipid metabolism]
domain (<xref ref-type="fig" rid="F1">Figure 1</xref>). However,
latter’s role in sterol desaturation is not clear
[<xref ref-type="bibr" rid="R23">23</xref>].
The <italic>EoChyb</italic> amino acid composition analysis revealed that it is rich
in alanine (A) amino acid (See Figure 1 <xref ref-type="supplementary-material" rid="SD1">supplementary material</xref>and Table 3 in
<xref ref-type="supplementary-material" rid="SD1">supplementary material</xref>).</p><p>The dendrogram constructed to study the phylogenetic relationship
between <italic>EoChyb</italic> and <italic>Chyb</italic> protein sequences from 13 dicots, 3 monocots
and 3 algal members shows 3 MC. The <italic>Chyb</italic> proteins from dicots,
monocots and algal members were precisely grouped in MC1, MC2, and
MC3 respectively (<xref ref-type="fig" rid="F2">Figure 2</xref>). All the <italic>Chyb</italic>proteins from dicotyledonous plants in MC1 were sub-grouped into 3 sub-clusters. The sub-cluster-1 is
of <italic>Chyb</italic> from <italic>Citrus sinensis</italic> and <italic>Glycine max</italic>. The sub-cluster-2 is of
<italic>Chyb</italic> from <italic>Coffea arabica</italic>, <italic>Solanum lycopersicum</italic>, <italic>Gentiana lutea</italic>,
<italic>Diospyros kaki</italic> and <italic>Capsicum annuum</italic>. Whereas, <italic>Vitis vinifera</italic>, <italic>Adonis
aestivalis</italic>, <italic>Chrysanthemum x morifolium</italic>, <italic>Brassica napus</italic>, <italic>Arabidopsis
thaliana</italic> and <italic>Daucus carota</italic> were grouped under sub-cluster-3 of MC1. All
the <italic>Chyb</italic> protein sequences from monocots, <italic>Crocus sativus</italic>, <italic>Elaeis
oleifera</italic>, <italic>Lilium hybrid</italic> and <italic>Zea mays</italic> used in phylogenetic analysis were
grouped under MC2; and three algal <italic>Chyb</italic> protein sequences from
<italic>Chlamydomonas reinhardtii</italic>, <italic>Muriella zofingiensis</italic> and <italic>Haematococcus
pluvialis</italic> were grouped under MC3 (<xref ref-type="fig" rid="F2">Figure 2</xref>).
This classification of <italic>Chyb</italic> is in line with the phylogenetic lineages
that have molecular data in the NCBI databases [<xref ref-type="bibr" rid="R24">24</xref>].
The dendrogram analysis also reflects that <italic>EoChyb</italic> is phylogenetically closer to
the <italic>Chyb</italic> from <italic>Lilium hybrid</italic> and other monocots used in the study.</p><p>The multiple sequence alignment of <italic>Chyb</italic> amino acid sequences of
<italic>EoChyb</italic> and <italic>Chyb</italic> protein sequences from 13 dicots, 3 monocots and 3
algal members (Table 2 see <xref ref-type="supplementary-material" rid="SD1">supplementary material</xref>) clearly shows the
well conserved catalytic residues in <italic>Chyb</italic>. The analysis of the multiple
sequence alignment of <italic>Chyb</italic> amino acid sequences clearly shows that in
total 51 amino acid residues are fully conserved in <italic>Chyb</italic> of the all plant
species and algal members (<xref ref-type="fig" rid="F3">Figure 3</xref>).
It was also evident that across the <italic>Chyb</italic> amino acid sequence, in total 28
amino acid residues were conserved strongly (<xref ref-type="fig" rid="F3">Figure 3</xref>).</p></sec><sec id="s5"><title>Conclusion</title><p>This study has annotated the salient features of randomly isolated <italic>EoChyb</italic>cDNA clone using Bioinformatics tools. Bioinformatics analyses revealed
that <italic>EoChyb</italic> protein is carrying conserved domains for the beta-carotene
hydroxylase and the sterol desaturase. Furthermore, the study also shows
the 51 fully conserved amino acid residues in <italic>Chyb</italic> from the flowering
(monocot and dicot) plants and algal members, which could be of
relevance to gain insights into the evolution of <italic>Chyb</italic>.</p></sec><sec sec-type="supplementary-material"><title>Supplementary material</title><supplementary-material content-type="local-data" id="SD1"><caption><title>Data 1</title></caption><media xlink:href="97320630005104S1.pdf" xlink:type="simple" id="d32e667" position="anchor" mimetype="application" mime-subtype="pdf"></media></supplementary-material></sec></body><back><ack><p>Authors are grateful to the Ministry of Science, Technology and
Innovation (MOSTI) of Malaysian Government for research funding
[Grant Code: IRPA: 01-02-02-0014/PR0015/07-07], and to the United
Plantation Berhad, Perak, Malaysia for supplying fruit samples of <italic>Elaeis
oleifera</italic> for this study.</p></ack><fn-group><fn id="FN1" fn-type="other"><p><bold>Citation:</bold>Bhore<italic>etal</italic>; Bioinformation 5(3): 104-112 (2010)</p></fn></fn-group><ref-list><title>References</title><ref id="R01"><label>1</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Muniran</surname><given-names>F</given-names></name><etal></etal></person-group><source>Indian J Exp Biol</source><year>2008</year><volume>46</volume><fpage>79</fpage><pub-id pub-id-type="pmid">18697576</pub-id></element-citation></ref><ref id="R02"><label>2</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Asemota</surname><given-names>O</given-names></name><name><surname>Shah</surname><given-names>FH</given-names></name></person-group><source>Afr J Biotechnol</source><year>2004</year><volume>11</volume><fpage>595</fpage></element-citation></ref><ref 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An open reading frame and noncoding
regions are shown in capital and small letters, respectively. The deduced amino acid sequence is given below the nucleotide sequence, which is numbered
at the both ends of each sequence line. The open reading frame encodes for a protein of 325 amino acid residues. Amino acid residues are numbered
beginning with the initial methionine till last glycine (G) residue. Initiation and termination codons are shown in bold. The beta-carotene hydroxylase
(fatty acid hydroxylase super family) conserved domain (PLN02601) [<xref ref-type="bibr" rid="R25">25</xref>] residues are shown in green colour. The sterol desaturase [Lipid metabolism]
domain (ERG3) [<xref ref-type="bibr" rid="R26">26</xref>] residues are underlined. *represent the termination codon. This cDNA was isolated by random method of gene isolation from <italic>E.
oleifera</italic> 17 week old mesocarp tissue cDNA library.</p></caption><graphic xlink:href="97320630005104F1"></graphic></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Rooted dendrogram showing clustering of beta-carotene hydroxylase (<italic>Chyb</italic>) from <italic>E. oleifera</italic> and other organisms. Amino acid sequences for
different organisms were obtained from NCBI database. Alignment of amino acid sequences and dendrogram construction was carried out using multiple
sequence alignment by ClustalW [<xref ref-type="bibr" rid="R19">19</xref>] program using default parameters. Location of <italic>E. oleifera Chyb</italic> in phylogenetic tree is shown in pink box. The ID
of <italic>Chyb</italic> proteins used in the study is given in Table 2 (see <xref ref-type="supplementary-material" rid="SD1">supplementary material</xref>). MC stands for main cluster.</p></caption><graphic xlink:href="97320630005104F2"></graphic></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Similarity comparison of amino acid sequences of the <italic>E. oleifera</italic> beta-carotene hydroxylase (<italic>Chyb</italic>) protein and <italic>Chyb</italic> amino acid sequences
from other organisms. Amino acid sequences are numbered at the end of each sequence row. (*), (:) and (.) denote single fully conserved residues,
residues with conserved strong groups and residues with conserved weak groups in <italic>Chyb</italic>, respectively. Aa, <italic>Adonis aestivalis</italic>; At, <italic>Arabidopsis thaliana</italic>;
Bn, <italic>Brassica napus</italic>; Can, <italic>Capsicum annuum</italic>; Car, <italic>Coffea arabica</italic>; Cr, <italic>Chlamydomonas reinhardtii</italic>; Csi, <italic>Citrus sinensis</italic>; Csa, <italic>Crocus sativus</italic>; Cxm,
<italic>Chrysanthemum x morifolium hybrid</italic>; Dc, <italic>Daucus carota</italic>; Dk, <italic>Diospyros kaki</italic>; Eo, <italic>Elaeis oleifera</italic>; Gl, <italic>Gentiana lutea</italic>; Gm, <italic>Glycine max</italic>; Hp,
<italic>Haematococcus pluvialis</italic>; Lh, <italic>Lilium hybrid</italic>; Mz, <italic>Muriella zofingiensis</italic>; Sl, <italic>Solanum lycopersicum</italic>; Vv, <italic>Vitis vinifera</italic>; Zm, <italic>Zea mays</italic>. This alignment is
produced by clustal 2.0.11 multiple sequence alignment program.</p></caption><graphic xlink:href="97320630005104F3"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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