碩士 / 臺灣大學 / 動物學研究所 / 98 / Anisakis simplex is a famous helminth parasite of commercial marine fish. Anisakiasis and allergy may occur when people ingest third stage larvae contained within the seafoods. The larvae in seafoods were usually killed by deep frozen. According to the former research, A. simplex could survive from freezing temperature and which threatened the safety seafood and fisheries industry. Organisms have evolved many antifreeze strategies for adaptation to low temperature. Trehalose accumulation during cold stress protects cells from damage and maintains the cellular metabolism, and the heat shock proteins are able to reactive the denature protein by refolding. In this study, we focus on the gene expression of trehalose synthase (TPS) and heat shock protein (HSP) of A. simplex under cold stress to investigate their role of antifreeze mechanism. The TPS gene sequences we amplified in this study have low similarity to the highly concerved domain between other species. Therefore, we use the predicted hsp and shsp (small heat shock pritein) sequence from A. simplex ESTs database to make functional analysis and design specific primers for Real-time PCR, then detect the gene expression under cold stress. Sequence analysis revealed that the hsp of A. simplex has the similar concerved functional domain to other small heat shock proteins. As the results, we suggested that it should belong to shsp family. shsp sequence analysis revealed that it does not have the similar concerved functional domain and the identity of with other shsp also low, so we suggested that the predicted shsp sequence may not be the sHSP gene. Real-time PCR results showed that the relative mRNA expression of hsp has a significant increase under cold stress, confirmed that the hsp was regulated by low temperature and the expression increase with the temperature cool-down. The gene expression of hsp at L3 larva stage is higher than L4 confirmed that the gene expression pattern is different with development stages. shsp in two low-temperature treatments and the two developmental stages were no significant increases shows it as not induced by low temperature. The phylogenetic tree of hsp show the similar evolution compare with the phylogenetic tree of mtDNA. This results confirm the opinion that hsp is highly conserved in the course on evolution of nematodes and other species.
| Identifer | oai:union.ndltd.org:TW/098NTU05312010 |
| Date | January 2010 |
| Creators | Yun-Chia Huang, 黃韻嘉 |
| Contributors | Hsiu-Hui Shih, 施秀惠 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 63 |
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