蝦類章魚胺生合成路徑功能之研究

甲殼類動物受到緊迫 (stress) 時會釋放生物胺，藉以調控生理及免疫反應。南美白蝦生物胺生合成機制主要分為兩個路徑，兩個合成路徑之前驅物皆為酪胺酸 (tyrosine, TR)。其一路徑為TR→L-多巴 (L-DOPA)→多巴胺 (dopamine, DA)→正腎上腺素 (norepinephrine, NE)，藉由酪胺酸羥化酶 (tyrosine hydroxylase, TH)、多巴脫羧酶 (dopa decarboxylase, DDC) 與多巴胺β羥化酶 (dopamine beta-hydroxylase, DBH) 所催化代謝合成，另一路徑為酪胺酸TR→酪胺 (Tyramine, TA)→章魚胺 (octopamine, OA)，藉由酪胺酸脫羧酶 (tyrosine decarboxylase, TDC) 與酪胺β羥化酶 (tyramine beta-hydroxylase, TBH) 催化代謝合成。本研究證實淡水長臂大蝦與草蝦分別注射 ≤ 250 pmol prawn−1與 ≤ 1000 pmol shrimp−1 OA後可顯著提升總血球數、不同血球數、酚氧化酵素活性、超氧陰離子活性、溶菌酶活性、葡萄糖與乳酸含量、對病原菌之吞噬作用與清除效率、及病原菌攻擊感染之活存率。草蝦注射 ≤ 10 nmol shrimp−1 OA可調節血球中Ca2+與cAMP含量。OA透過α和β腎上腺素受體的潛在細胞訊息傳遞路徑調節淡水長臂大蝦免疫能力。自南美白蝦腦中選殖TDC基因序列全長 (2724 b.p.)，可轉譯區為2337 b.p.且可轉譯成779個胺基酸的蛋白質序列，具高度保守之Pyridoxal_deC domain且大量表現在胸神經中。RNAi基因靜默LvTDC後會顯著降低透明血球數、超氧陰離子活性、吞噬活性、清除效率及抗菌生肽ALF的基因表現量，進而降低對溶藻弧菌的抗病能力。自南美白蝦腦中選殖TBH基因序列全長2178 b.p.，可轉譯區為1227 b.p.且可轉譯成408個胺基酸的蛋白質序列，具兩個高度保守的Cu2_monooxygen domain，並在血球與其他神經組織中 (包括腦、胸神經節、腹部神經節和圍咽神經) 皆有表現，當暴露於低溫緊迫或病原菌感染時，血球與大腦中的LvTBH表現量顯著升高，進而導致血淋巴中的OA含量會隨之升高，證實LvTBH參與在壓力緊迫時南美白蝦神經內分泌的調節。以上研究結果證實OA生物胺生合成路徑產物及其關鍵酵素參與在蝦類生理及免疫抗病能力之調節。

關鍵字

章魚胺；淡水長臂大蝦；草蝦；南美白蝦；酪胺酸脫羧酶；酪胺β羥化酶；神經內分泌；免疫抗病能力

並列摘要

Bioamines (BAs) play critical roles in regulating physiological and immunological homeostasis in crustacean under stressful environments. The BAs biosynthesis pathway divided into two pathways, and the precursor of both synthetic pathways is tyrosine (TR). One of the BAs synthesis pathway is TR→L-DOPA→dopamine→norepinephrine, and catalyze by tyrosine hydroxylase, dopa decarboxylase and dopamine beta-hydroxylase. The other BAs synthesis pathway is TR→tyramine (TA)→octopamine (OA), and catalyze by tyrosine decarboxylase (TDC) and tyramine beta-hydroxylase (TBH). In the present study documented that Macrobrachium rosenbergii and Penaeus monodon which had received OA at ≤ 250 pmol prawn−1 and ≤ 1000 pmol shrimp−1 showed upregulation of total haemocyte count, differential haemocyte count, phenoloxidase activity, respiratory bursts, superoxide, phagocytic activity and clearance efficiency in response to the pathogen and survival rate in challenge test. Pharmacological actions of antagonists against α- and β- adrenergic receptors had been used to evaluate the potential modulating pathway of M. rosenbergii. OA administration by injection at ≤ 10 nmol shrimp−1 mediates transient upregulation of intracellular Ca2+ and cAMP second messenger pathways of P. monodon. TDC and TBH are key enzymes in the biosynthesis pathway of TR→TA→OA. Full-length cDNA of L. vannamei TDC (LvTDC) gene was cloned which contained an open reading frame (ORF) of 2337 b.p. and was predicted to encode a protein of 779 amino acids. LvTDC has a highly conserved Pyridoxal_deC domain and was abundantly expressed in the thoracic ganglia. The hyaline cell, respiratory bursts, phagocytic activity and clearance efficiency in response to V. alginolyticus and the gene expression of antimicrobial peptide ALF were significantly decreased through the LvTDC silencing. A full-length cDNA of TBH was cloned and characterized from L. vannamei (LvTBH) and its expression in haemocytes and nervous tissue including brain, thoracic ganglia, abdominal ganglia and circumoesphageal connective, which comprised 2178 nucleotide residues and contained an ORF encoding 408 amino acids and has two highly conserved Cu2_monooxygen domains. LvTBH gene expression in haemocyte and brain were up-regulation when exposed to hypothermal stress and V. alginolyticus infection as OA level increased in haemolymph. These results suggest that LvTBH possess a conserved function in neuroendocrine regulatory network of shrimp. It confirms that the bioamines and key enzymes in TR→TA→OA biosynthetic pathway participate in the physiological and immunological functions of L. vannamei.

並列關鍵字

octopamine ； Macrobrachium rosenbergii ； Penaeus monodon ； Litopenaeus vannamei ； tyrosine decarboxylase ； tyramine beta-hydroxylase ； neuroendocrine ； immune response and disease resistance

參考文獻

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