Genetic diversity of selected commercial freshwater fishes based on phospholipase C zeta expression and muscle protein profiling
Egg activation is important to help releases the egg from meiotic arrest and blocks polyspermy. It is linked with an increase in intracellular egg calcium ions (Ca2+) in almost all species studied and current studies imply that the mammalian sperm factor involved is a sperm-specific phospholipase...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2014
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/70446/1/FPSK%28M%29%202014%2056%20-%20IR.pdf |
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| Summary: | Egg activation is important to help releases the egg from meiotic arrest and blocks
polyspermy. It is linked with an increase in intracellular egg calcium ions (Ca2+) in
almost all species studied and current studies imply that the mammalian sperm factor
involved is a sperm-specific phospholipase C zeta, PLCζ. Here, we first reported the
identification of PLCζ in the testis and egg of Lampam Jawa. Our findings provide
the evidence that PLCζ is present in the species of male and female Lampam Jawa
(Barbonymus gonionotus). For this study, six types of commercial freshwater fish
were selected i.e. Red Tilapia (Oreochromis sp. Red Tilapia), Black Tilapia
(Oreochromis mossambicus), Catfish or Keli (Ictalurus punctatus), Silver Catfish or
Patin (Pangasius pangasius), Snakehead Fish or Haruan (Channa striata) and Silver
Barb or Lampam Jawa (Barbonymus gonionotus). The objectives of this study were
to isolate the mRNA from the gonads of freshwater fishes, to identify and amplify
the phospholipase C zeta (PLCζ) gene fragments, to sequence the purified DNA
fragments and to compare the PLCζ sequence to other PLCζ sequence available in
NCBI database, to characterize muscle protein of selected commercial freshwater
fish and lastly, to compare phylogenetic trees of 16S rDNA generated. In addition,
protein profiles can be used as indicators of evolutionary relatedness. The differences
and similarity aspects of fish muscle protein were measured and the relatedness
based on protein profile was compared with the relatedness of fishes obtained from
16S rDNA sequences alignment by using dendrogram. The methods used for the
expression of PLCζ were RNA extraction, spectrophotometric quantitation of RNA,
Two-Step RT-PCR reaction, agarose gel electrophoresis, gel documentation, gel
extraction, sequencing and dendrogram. The methods used for muscle protein
profiling were sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE),
gel viewing, muscle’s protein banding profile analysis, genetic diversity and
lastly comparison of protein profile and 16S phylogenetic tree generated by using
dendrogram. For the study of PLCζ expression, male and female Lampam Jawa
showed bands at around 420bp in agarose gel electrophoresis that indicated the presence of PLCζ gene and no significant bands were found in other types of fishes
used in this study. For muscle protein profiling, the multiple bands of proteins
obtained from SDS – PAGE showed similar protein contents among different fish
species used in this study. The dendrogram showed the highest percentage of
similarity is between Tilapia Hitam and Tilapia Merah which is 84% followed by
Haruan and Patin which exhibited less than 84% similarity. Keli had 67% similarity
with Haruan, Patin, Tilapia Merah and Tilapia Hitam while Lampam Jawa showed
less than 60% similarity with Keli, Patin, Haruan, Tilapia Merah and Tilapia Hitam. |
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