Molecular cloning, characterization and expression analysis of prophenoloxidase gene in longlegged spiny lobster Panulirus longipes

Abstract

White shrimp Litopenaeus vannamei receiving fucoidan at 2, 6, and 10 μg g−1 after 0–144 h or 0–120 h were examined for immune parameters (haemograms, phenoloxidase activity, respiratory burst, and superoxide dismutase activity), proliferation of haemocyte in the haematopoietic tissue (HPT), geneexpression, and phagocytic activity and clearance efficiency to Vibrio alginolyticus. Immune parameters and mitotic index of HPT increased after 3–24 h, reached their maxima after 48–72 h, and returned to background values after 144 h. Transcripts of lipopolysaccharide and β-1,3-glucan binding protein (LGBP), peroxinectin (PX), prophenoloxidase (proPO) I, proPO II, astakine, and haemocyte homeostasis-associated protein (HHAP) were up-regulated to a maximum after 48–72 h and returned to background values after 144 h. Phagocytic activity and clearance efficiency to V. alginolyticus increased after 12 h, reached its maximum after 48 h, and continued to remain higher after 120 h. In another experiment, shrimp receiving fucoidan after 48 h and 144 h were respectively challenged with V. alinolyticus at 6 × 106 colony-forming units (cfu) shrimp−1 or challenged with WSSV at 1.2 × 105 copies shrimp−1 and then placed in seawater. The survival rate of shrimp receiving fucoidan was significantly higher than in controls.Inconclusion, shrimp receiving fucoidan showed a proliferation of HPT, increased immune parameters, and up-regulated transcripts of LGBP, PX, proPO I, proPO II, astakine, and HHAP after 48 h. Shrimp receiving fucoidan exhibited a defense against V. alginolyticus and WSSV, even after immune parameters recovered to background levels.以反轉錄－酵素聚合酶反應（RT-PCR），3'端與5'端序列快速增幅（RACE）技術選殖長足龍蝦（Panulirus longipes）原酚氧化酵素cDNA全長序列，其序列全長為2993 bp，包含一段2010 bp開放式讀框、58 bp的5'端非轉譯區，以及一段包含poly A訊息的925 bp 3'非轉譯區。開放式讀框可轉錄一段670個胺基酸的蛋白質片段，推估分子量為76.23 kDa，推估等電點為5.60。蛋白質序列內有兩個酪氨酸酶銅結合區，被6個組胺酸包圍（copper A，193，197，219，copper B，353，357，393），本原酚氧化酵素序列上有一段類硫酯鍵序列（GCGWPQHV），此序列結構上與其他的甲殼類原酚氧化酵素上的序列相似。本原酚氧化酵素序列亦包含了3個可能進行醣基化的位置（NES，NTT，NVN），以及一段保守的C-端序列，此C-端序列與其它原酚氧化酵素已知序列的相似。序列比較，顯示長足龍蝦原酚氧化酵素與螯龍蝦、對蝦、淡水蝦、螯蝦以及昆蟲的原酚氧化酵素序列相似度分別為75％、69％～72％、67％～68％、65％～68％、60％〜68％以及49％～53％。長足龍蝦注射glucan後120小時，原酚氧化酵素基因轉錄明顯增加。