摘要
海獸胃線蟲(Anisakis simplex)是海洋經濟魚類常見之寄生性蠕蟲，人類若食用含有其第三期幼蟲之海鮮則可能罹患海獸胃線蟲症或過敏症。漁產品主要以冷凍方式殺死此寄生蟲，然而前人研究顯示此蟲對低溫具有耐受性，因而成為影響食品安全與漁業經濟之一大隱憂。為了適應低溫逆境，生物體演化出許多不同的抗凍機制。前人研究證實，低溫下海藻糖的累積有助於生物體降低凝固點及維持細胞正常生理作用；而熱休克蛋白能幫助在逆境下變性失活的蛋白質重新摺疊。因此本研究針對海獸胃線蟲之海藻糖生成酶(TPS)與熱休克蛋白(HSP)基因在低溫逆境下的表現，探討兩者是否參與海獸胃線蟲之抗凍機制。由於增幅TPS基因的過程中並未獲得可信賴之序列片段，因此改以海獸胃線蟲ESTs database中所預測之hsp與shsp序列片段進行分析，並設計專一性引子以Real-time PCR偵測兩者在低溫下mRNA的相對表現量。序列分析結果顯示海獸胃線蟲hsp片段具有與微小熱休克蛋白相似之保守性功能區，且和其他物種之shsp序列有高相似性，故推論其應屬於shsp家族。海獸胃線蟲之shsp序列分析結果顯示其不具有保守性功能區，與其他物種之shsp相似性也偏低，因此推測該片段可能並非sHSP基因。Real-time PCR 結果顯示hsp之mRNA相對表現量在低溫誘導下有顯著增加，證實該基因受低溫的調控，而且溫度越低表現量越高。海獸胃線蟲第三期幼蟲之hsp表現量高於第四期幼蟲，證實不同發育階段基因表現的模式亦不相同。 shsp基因在兩種低溫環境及兩種發育階段中都無顯著表現，顯示其不受低溫所誘導。以hsp蛋白序列建立親緣樹並與相對保守之mtDNA親緣樹進行比較，結果顯示兩者演化方向吻合，證實hsp在線蟲及其他物種的演化上都具有高度保守性。
並列摘要
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.