Optimization Of RNA Extraction And Generation Of Expressed Sequence Tags From Sargassum Binderi (Sonder) J. Agardh
Brown seaweeds are macroalgae in the marine habitat. They are widely used as food, sources for cosmetics, pharmaceuticals, soap, agar, textile, and alginate. Sargassum binderi is one of the most abundant brown seaweeds in Malaysia. Previous studies have shown that S. binderi contains high qualit...
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| Format: | Thesis |
| Language: | English |
| Published: |
2005
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/5926/1/FBSB_2005_15%20IR.pdf |
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| Summary: | Brown seaweeds are macroalgae in the marine habitat. They are widely
used as food, sources for cosmetics, pharmaceuticals, soap, agar, textile,
and alginate. Sargassum binderi is one of the most abundant brown
seaweeds in Malaysia. Previous studies have shown that S. binderi contains
high quality of alginate compared to commercially used seaweeds such as
Laminaria hyberborea, Macrocystis pyrifera and Ascophyllum nodosum.
Expressed sequence tag (EST) approach is a powerful tool in providing
genetic information of an organism, especially for S. benderi in which little
genetic information is available. This study presents the first attempt in
generating ESTs from S. binden. RNA extraction from seaweeds was the
main challenge in this study, as the RNA yield was low and the
polysaccharide contamination was difficult to be eliminated. A total of ten
different RNA extraction methods (including five modifications) have been
carried out to obtain sufficient RNA of high quality to construct a
representative cDNA library for ESTs generation. The optimized S. binderispecific
CTAB RNA extraction method developed in this study was able to
produce high yield of RNA with minimum polysaccharide contamination.
Sufficient amount of mRNA was obtained to construct a primary cDNA library
with a titer of 9.2 x lo5 pfulml. A total of 2051 ESTs were generated and
analyzed from the amplified cDNA library of S. binden (with a titer of 1.31 x
lo9 pfulml). The ESTs were putatively identified by comparison to the nonredundant
peptide database in NCBI. Approximately 82% of the ESTs were
assigned as unknown and novel sequences that are potentially important for
new gene discovery, whereas the 18% ESTs with significant matches to the
database were classified into various putative functional groups, including
protein synthesis, energy, protein destination and storage, metabolism, cell
structurel division, diseasel defense, signal transduction, transcription, and
miscellaneous. The EST information generated from this study may
contribute towards better understanding of the biochemistry and molecular
biology of S. binder; and other brown seaweeds in the future. |
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