Isolation, characterization and mapping of expressed sequence tags (ESTs) from pineapple fruit cDNA library

Ong , Wen Dee (2011) Isolation, characterization and mapping of expressed sequence tags (ESTs) from pineapple fruit cDNA library. Masters thesis, Universiti Malaysia Sabah.

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Abstract

Pineapple (Ananas comosus var. comosus), is an important tropical non-climacteric fruit with high commercial potential. Understanding the phenomena behind fruit ripening with a focus on improving fruit quality traits such as flavor, texture, appearance and sweetness may be possible through gene expression profiling of pineapple fruit transcriptome. As such, the objectives of this project are to, firstly, construct and sequenced mature green pineapple cDNA and de novo assembly of paired-end Solexa reads. Secondly, to characterize and functionally annotate the transcripts through similarity search and mapping against Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database respectively. Finally, to develop a database of Expressed Sequence Tags containing Simple Sequence Repeats (EST-SSRs) using the newly obtained transcripts and/or through pineapple ESTs that are available in GenBank. The results show that both the unique transcripts (UT) assembled pineapple sequences and contigs from de novo assembly generated a total of 28,896 transcripts being generated with length ranges from 100 bp to 3.8 kb. A search for sequence similarity with NCBI's non redundant database identified about 17,049 transcripts which were found to be associated with primary metabolisms, amino acid synthesis and processing, membrane and transport, cell division, cytoskeleton, cell wall and metabolism, RNA related gene expression, signal transduction, defense and stress related protein and also secondary metabolisms. Out of these transcripts, 71% returned GO terms with the distribution among the ontologies given as such: 35.8% in molecular function, 33.5% in cellular component and 30.7% in biological process. Annotation against the KEGG database pathways on the other hand, enabled the assignment of 542 enzyme commissions to 13,598 transcripts. The enzymes were further categorized into a total of 126 pathways with 122 pathways being involved in pineapple metabolism. The metabolic and cellular processes points out that there are tremendous changes in metabolic activities during pineapple fruit maturation as seen by the large numbers of the annotated transcripts. Data mining of the pineapple transcripts EST-SSRs showed that only 4% of the pineapple transcripts contained SSRs. Dinucleotide SSR (49.5%) was the most abundant followed by trinucleotide SSR (46.8%). The least abundant was tetranucleotide SSR (3.7%). Out of these, about 40% of the pineapple transcripts were found to have suitable flanking sites to enable the design of the upstream and downstream primers for future PCR amplification. This research cataloged the first pineapple fruit transcriptome. The transcripts will be subsequently useful to develop microarray chips for future gene expression studies among different plant tissue and development stages of the fruit. Further validation and/or relevant use of the ESTSSRs found will be useful in comparative mapping and genome mapping and gene tagging in pineapple.

Item Type:Thesis (Masters)
Uncontrolled Keywords:tropical non-climacteric fruit, pineapple, Gene Ontology, EST-SSRs, mapping
Subjects:Q Science > QK Botany
Divisions:SCHOOL > Biotechnology Research Institute (BRI)
ID Code:6652
Deposited By:IR Admin
Deposited On:30 Jul 2013 12:25
Last Modified:30 Jul 2013 12:25

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