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Molecular Characterization and Expression Profiling of Genes Involved in Ripening Banana

Lokesh, V. (2015) Molecular Characterization and Expression Profiling of Genes Involved in Ripening Banana. PhD thesis, University of Mysore.

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Abstract

Though phytohormones and signaling compounds are known to play important roles in ripening, exact mechanisms through which these molecules are involved in ripening physiology are not completely known except for the involvement of the phytohormone - ethylene. As climacteric ripening is associated with hundreds of genes that are regulated by many factors, differential display RTPCR (DDRTPCR) coupled with silver staining was performed to identify some of the differentially regulated genes by the phytohormones (ethylene, gibberellic acid, indole-acetic acid, abscisic acid and kinetin) and signaling compounds (salicylic acid and nitric oxide). Using 10 primer combinations and 40 populations of mRNA (5 stages of each treatments including control), a total of 68 bands were obtained, of which 52 were cloned into T/A cloning vector and sequenced. A total of 32 differentially regulated genes were identified after removing all the redundant and false positive sequences. These 32 sequences were identified, based on homology search using NCBI-BLAST as well as BLAST search in banana genome database. All the sequences were submitted to NCBI-EST library and the accession numbers were obtained. The sequences were categorized into groups based on their putative functions, such as in stress and senescence, in transcription and translation related, transport, carbohydrate metabolism and some with unknown functionalities. In addition to these reported proteins, several unknown gene fragments were also identified for the first time in ripening fruits of NR. The differential expression of these genes was further confirmed by quantitative real time PCR to know the exact extent of change in expression level. The identified differentially expressed genes could be further explored for their actual roles in ripening in relation to phytohormones and signaling compounds. The information generated through experiments of this chapter are useful in elucidating the role of the above- mentioned phytohormones and signaling compounds in ripening. The identification of full length gene sequence especially the coding sequence (CDS) is a crucial step in the functional annotation of genes. CDS is a sequence of nucleotides that corresponds to a specific sequence of amino acids in a protein. Apart from CDS, the other non-coding UTR regions at 5’ and 3’ end of the have also been of great significance owing to their significance in the regulation of the corresponding Abstract gene expression. From the 32 genes that were differentially regulated under the influence of various phytohormones and signaling compounds, three genes viz., 1- aminocyclopropane carboxylate oxidase (ACO), probable galacturonosyltransferase 7 (GAUT-7) and ubiquitin which were of great relevance either to fruit ripening or to phytohormone signaling were amplified for full length gene sequence using RACE approach. ACO full length gene sequence was 1159 with 957 bp CDS. GAUT-7 full length gene sequence was 2156 bp with 1359 bp CDS and the other full length gene coding for ubiquitin was 448 bp and the ORF region was of 234 bp. All the genes were analyzed for their transcription start site, miRNA target sites at 3’ UTR using several online tools. The gene sequences were also checked for their similarity with the corresponding AAA group banana gene sequences and were highly similar to the AAA group gene sequences. The prime task of shelf life extension for tropical climacteric fruits such as banana relies on the suppression of ethylene through controlled elicitation of certain metabolic regulators, which in turn counter ethylene formation. NO’s role in delaying/ inhibiting fruit ripening has been of great interest and many delivery systems are being practiced. Various NO donors were screened for their efficacies in releasing NO in a desirable manner for enhancing fruit attributes. Among several NO donors, SNP at 1 mM concentration appeared beneficial and hence was further analyzed for its effects on the nutritional profile of banana fruits. While the nutrition profiles were not affected, the SNP treatment enhanced fruit quality by enhancing shelflife and inhibiting phenylpropanoid pathway enzymes like PAL and PPO. Among many proposed mechanisms of NO regulation in plants, information on polyamine (PA)-mediated regulation is poor. Owing to both NO and PA’s role in fruit ripening control, the effect of NO in altering PAs level and the resulting modulation in ethylene level was analyzed by studying the alterations in PA levels after SNP treatment.. It was found that putrescine content increased by 5.8 and 9 fold in pulp and peel respectively. Spermine was induced more in peel (10.2 folds). Spermidine levels were induced by 2.9 and 8.3 folds in pulp and peel of Cavendish banana respectively. The expression studies of ethylene pathway genes ACS and ACO showed that they were downregulated in both Cavendish and NR fruits. ACS was downregulated by 5.2 and 7.2 folds on the 5th day. The other ethylene pathway gene ACO was also downregulated by 3.1 and 4.5 folds in Cavendish and NR banana Abstract fruits respectively. PA pathway genes were found upregulated in at least few stages of ripening after the SNP treatment. ODC was found upregulated by 3.8 and 2.3 folds in Cavendish and NR banana fruits respectively. SAMdC was upregulated by around 8 fold in Cavendish and SPMSYN and SPDSYN were also found consistently upregulated by SNP treatment. However, ADC was the gene which recorded the maximum upregulation by 14.93 and 10.4 folds in Cavendish and NR banana fruits respectively thus suggesting induction in putrescine levels by NO occurs via agmatine by up-regulation of ADC and probably not much via ornithine and ODC. Effect of various phytohormones and signaling compounds (Gibberellic acid, Auxin, Cytokinin, Abscisic acid, Salicylic acid, Methyl jasmonate, and Nitric oxide) for inhibiting/ delaying ripening was evaluated. Expression pattern of genes involved in diverse class of biochemical and physiological functions like cell wall hydrolysis (PG, PL, PME, β-galacturonase, Exp1 and Exp2), carbohydrate metabolism (Sucrose synthase, SPS, α-amylase and β-amylase), ethylene pathway (ACS and ACO), ethylene signal perception and transduction (ERS1, ERS2, EIL1, EIL2, EIL3, EIL4, MADS1, MADS2, MADS4 and MADS6), senescence and defense (CAT, SOD, PAL and chitinase) was studied. Though the maximum inhibition of ripening was imparted by gibberellic acid, it could not down-regulate all class of genes. Diverse effect was imparted by different growth regulators on various classes of ripening-related genes. Based on genetic expression data, various formulations of these phytohormones and signaling compounds were developed and were analyzed for their efficiency to delay fruit ripening.

Item Type: Thesis (PhD)
Uncontrolled Keywords: banana, ripening, genes profiling, phytohormones
Subjects: 500 Natural Sciences and Mathematics > 07 Life Sciences > 03 Biochemistry & Molecular Biology
600 Technology > 08 Food technology > 24 Fruits > 02 Banana
Divisions: Plant Cell Biotechnology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 18 May 2016 12:41
Last Modified: 18 May 2016 12:41
URI: http://ir.cftri.com/id/eprint/12168

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