21 research outputs found

    Characterization of an AGAMOUS-like MADS Box Protein, a Probable Constituent of Flowering and Fruit Ripening Regulatory System in Banana

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    <div><p>The MADS-box family of genes has been shown to play a significant role in the development of reproductive organs, including dry and fleshy fruits. In this study, the molecular properties of an AGAMOUS like MADS box transcription factor in banana cultivar Giant governor <em>(Musa sp</em>, AAA group, subgroup Cavendish) has been elucidated. We have detected a CArG-box sequence binding AGAMOUS MADS-box protein in banana flower and fruit nuclear extracts in DNA-protein interaction assays. The protein fraction in the DNA-protein complex was analyzed by mass spectrometry and using this information we have obtained the full length cDNA of the corresponding protein. The deduced protein sequence showed āˆ¼95% amino acid sequence homology with MA-MADS5, a MADS-box protein described previously from banana. We have characterized the domains of the identified AGAMOUS MADS-box protein involved in DNA binding and homodimer formation <em>in vitro</em> using full-length and truncated versions of affinity purified recombinant proteins. Furthermore, in order to gain insight about how DNA bending is achieved by this MADS-box factor, we performed circular permutation and phasing analysis using the wild type recombinant protein. The AGAMOUS MADS-box protein identified in this study has been found to predominantly accumulate in the climacteric fruit pulp and also in female flower ovary. <em>In vivo</em> and <em>in vitro</em> assays have revealed specific binding of the identified AGAMOUS MADS-box protein to CArG-box sequence in the promoters of major ripening genes in banana fruit. Overall, the expression patterns of this MADS-box protein in banana female flower ovary and during various phases of fruit ripening along with the interaction of the protein to the CArG-box sequence in the promoters of major ripening genes lead to interesting assumption about the possible involvement of this AGAMOUS MADS-box factor in banana fruit ripening and floral reproductive organ development.</p> </div

    Wet lab validation of <i>in silico</i> designed genic-SSR markers of mango.

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    <p><b>a)</b> PCR results with 48 different SSR markers (MSSR1- MSSR 48) in mango variety Amrapali; <b>b)</b> Allelic polymorphism of MSSR-13 in 8 different varieties of Mango 1. Neelam, 2. Dashehari, 3. Amrapali, 4. Chausa, 5. Pusa Lalima, 6. Ratual, 7. Mallika and 8. Alphonso.</p

    Leaf Transcriptome Sequencing for Identifying Genic-SSR Markers and SNP Heterozygosity in Crossbred Mango Variety ā€˜Amrapaliā€™ (<i>Mangifera indica</i> L.)

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    <div><p>Mango (<i>Mangifera indica</i> L.) is called ā€œking of fruitsā€ due to its sweetness, richness of taste, diversity, large production volume and a variety of end usage. Despite its huge economic importance genomic resources in mango are scarce and genetics of useful horticultural traits are poorly understood. Here we generated deep coverage leaf RNA sequence data for mango parental varieties ā€˜Neelamā€™, ā€˜Dashehariā€™ and their hybrid ā€˜Amrapaliā€™ using next generation sequencing technologies. De-novo sequence assembly generated 27,528, 20,771 and 35,182 transcripts for the three genotypes, respectively. The transcripts were further assembled into a non-redundant set of 70,057 unigenes that were used for SSR and SNP identification and annotation. Total 5,465 SSR loci were identified in 4,912 unigenes with 288 type I SSR (n ā‰„ 20 bp). One hundred type I SSR markers were randomly selected of which 43 yielded PCR amplicons of expected size in the first round of validation and were designated as validated genic-SSR markers. Further, 22,306 SNPs were identified by aligning high quality sequence reads of the three mango varieties to the reference unigene set, revealing significantly enhanced SNP heterozygosity in the hybrid Amrapali. The present study on leaf RNA sequencing of mango varieties and their hybrid provides useful genomic resource for genetic improvement of mango.</p></div

    Analysis of expression of MA-MADS5 protein in various regions of banana fruit tissue. a

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    <p>Various regions of banana fruit from the cultivar Giant governor (<i>Musa sp</i>, AAA group, subgroup Cavendish) have been indicated (zones Aā€“E). <b>b</b> Detection of MA-MADS5 protein in A and B zones of preclimacteric (0 DAH), climacteric (88 DAH) and postclimacteric (92 DAH) banana fruit by immunoblotting using anti-MA-MADS5 polyclonal antibody (1āˆ¶1000 dilution) (upper panel). Equal amounts of small nuclear protein was loaded in each lane and shown as loading control (lower panel). <b>cā€“e</b> Immunodetection of MA-MADS5 protein levels in C, D and E zones (including both peripheral and central parts) of preclimacteric, climacteric and postclimacteric fruit pulp tissues of banana (upper panel). Equal amounts of small nuclear protein was loaded in each lane and shown as loading control (lower panels of c, d and e respectively). Representative images from at least three independent experiments are shown for Figure bā€“e. <b>f and g</b> Immunolocalization of MA-MADS5 protein in A and B zones in postclimacteric banana pulp tissues (92 DAH). The legends of the panels Iā€“IV are similar as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044361#pone-0044361-g005" target="_blank">Figure.5</a> bā€“e.</p

    Detection of CArG-box DNA binding protein in banana fruit and flower nuclear extracts.

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    <p><b>a</b> CArG box motif, derived from <i>Arabidopsis</i> Agamous MADS box binding site, was used as probe for gel mobility shift assays. The MADS domain consensus binding site (CArG box motif) has been indicated (italics and underlined). DNA fragment containing the modified CArG box motif (highly conserved T and A residues were changed to G and C residues) has been indicated (small letters underlined). <b>b</b> Gel mobility shift assay using labeled CArG-box DNA as probe (A probe). Lane 1 contained only radiolabeled CArG-box DNA probe, while 15 Āµg nuclear protein extract was added in lanes 2ā€“10. Lane 2 to 5 contained nuclear protein extract from banana flower and fruit pulp tissues at the preclimacteric, climacteric and postclimacteric stages of ripening with labeled CArG box motif as probe. Lane 6 and 7 contained nuclear protein extract from banana flower and climacteric fruit pulp with labeled modified CArG box motif as probe (Am probe). In lane 8 and 9, nuclear protein extract from banana flower and climacteric pulp was incubated with labeled CArG box motif in presence of 100-fold excess of unlabeled (non-radioactive) CArG box motif. Lane 10 contained nuclear protein extract from climacteric banana pulp with labeled CArG box motif in presence of 100-fold excess of unlabeled modified CArG box motif. Com-competitor, NE-nuclear extract. <b>c</b> South-Western blot analysis with nuclear protein extracts isolated from flower and climacteric banana fruit pulp (lanes 1ā€“2). āˆ¼25 Āµg of nuclear extract was loaded in each lane. The radiolabeled synthetic oligo containing CArG box motif was used as probe. <b>d</b> Equal amounts of small nuclear protein (SNP) from banana flower and climacteric pulp tissues were resolved in 12% SDS-PAGE and has been shown as loading control. <b>e</b> Measurements of GUS activity in transgenic tobacco lines carrying trimeric CArG-box motif (3X CArG) or the modified trimeric CArG-box sequence (m) in fusion with <i>GUS</i>. GUS activity was detected in the leaves of control and transgenic tobacco lines. The error bars indicate mean values from three independent observations. <b>f</b> Identification of 27-kDa CArG-box binding MADS-domain protein by mass spectrometry. Overall sequence coverage of the peptides with the matched protein (Q4TTS9_MUSAC of <i>Musa acuminata</i>). Matched peptides shown in red letters. Experiments were repeated three times. Representative images from at least three independent experiments are shown for Figure bā€“d.</p
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