- 學位論文
白蝦O型乙醯葡萄糖胺轉移酶基因分子轉殖與特性之分析
Molecular cloning and characterization of O-GlcNAc transferase gene in white shrimp, Litopenaeus vannamei
摘要
本研究從白蝦 (Litopenaeus vannamei)肝胰腺中選殖O型乙醯葡萄糖胺轉移酶 (O-GlcNAc transferase)全長基因，並利用即時定量聚合酶連鎖反應觀察其在眼柄、肌肉、表皮、神經節、血淋巴、鰓、胃、腸道及肝胰腺的表現，並分析白蝦受病原菌感染後O型乙醯葡萄糖胺轉移酶的表現。O型乙醯葡萄糖胺轉移酶全長cDNA為3,275 bp，包含2,988 bp之開放閱讀區、174 bp的5’ 端非轉譯區和113 bp的3’ 端非轉譯區，可轉譯出996個胺基酸，包含12個三十四肽重複性序列 (Tetratricopeptide repeats, TPRs)，推測分子量為111.34 kDa，估計等電點為6.50。演化樹分析發現，O型乙醯葡萄糖胺轉移酶在蝦類、魚類和哺乳動物之間具有高度的保守性。O型乙醯葡萄糖胺轉移酶在所有檢測的組織中皆有表達，其中以血球、鰓及腸道表現量較高。此外，白蝦有攝食Bacillus subtilis E20的情況下，O型乙醯葡萄糖胺轉移酶及熱休克蛋白70的基因和O型乙醯葡萄糖胺轉移酶及熱休克轉錄因子1的蛋白質表現皆顯著高於控制組；另外，白蝦受弧菌感染後，O型乙醯葡萄糖胺轉移酶及熱休克蛋白70的基因和O型乙醯葡萄糖胺轉移酶及熱休克轉錄因子1的蛋白質表現在白蝦受溶藻弧菌感染後顯著高於注射生理鹽水組。因此，推測白蝦攝食B. subtilis E20後可以提升O型乙醯葡萄糖胺轉移酶的表現，進而促進白蝦受病原感染後的免疫調節。
並列摘要
In the present study, the full-length O-GlcNAc transferase (OGT) cDNA was cloned from the hepatopancreas of white shrimp (Litopenaeus vannamei). Its expression in eyestalk, muscle, skin, nerve, haemocytes, gill, stomach, intestine and hepatopancreas was also investigated through the real-time PCR. In addition, the gene expression of OGT after pathogen infection was analyzed. The full-length cDNA of OGT was 3,275 bp, including an open reading frame (ORF) of 2,988 bp, a 174 bp 5’ untranslated terminal region (UTR), and a 113 bp 3’ UTR, which encoding a protein of 996 amino acids, and containing 12 tetratricopeptide repeats (TPRs). The calculated molecular mass of protein is 111.34 kDa with an estimated pI of 6.50. The highly conservation of OGT gene among shrimp, fish and mammals was also observed through phylogenetic analysis. OGT gene was ubiquitously expressed in all detected tissues with highest expressions in haemocytes, gill and intestine. The gene expressions of OGT and heat shock protein 70 (HSP70), and the protein concentrations of OGT and heat sock factor 1 (HSF-1)were significantly increased in shrimp fed with B. subtilis E20 as compared to control. Moreover, the gene expressions of OGT and HSP70, and the protein concentrations of OGT and HSF-1 were significantly increased after shrimp being challenged with Vibrio alginolyticus compared to shrimp injected with saline. It is considered that B. subtilis E20 might positive effect on the OGT expression after pathogen infection resulting in the improvement of shrimp health status.