Molecular Cloning, Characterization and Expression Analysis of Prophenoloxidase Gene in Longlegged Spiny Lobster Panulirus longipes

長足龍蝦（棘龍蝦）原酚氧化酵素基因選殖，特徵及表現

林永慶(Yong-Chin Lin) ； Baskaralingam Vaseeharan ； 柯琦峰(Chi-Fong Ko) ； 黃千倫(Chien-Lun Huang) ； 陳建初(Jiann-Chu Chen)

《臺灣水產學會刊》 43卷3期 (2016/09) Pp. 179-194

https://doi.org/10.29822/JFST.201609_43(3).0005

摘要

A prophenoloxidase (proPO) cDNA was cloned from the hemocytes of longlegged spiny lobster Panulirus longipes using oligonucleotide primers and RT-PCR. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end (RACE) method. Analysis of the nucleotide sequence revealed that the cDNA clone has a full-length of 2993 bp, with an open reading frame of 2010 bp, a 58-bp 5'-untranslated region, and a 925-bp 3'-untranslated region containing a poly A signal. It encodes a protein of 670 amino acids with a predicted molecular weight of 76.23 kda and with an estimated pI of 5.60. It contains two putative tyrosinase copper binding motifs with six histidine residues (copper A, 193, 197, 219, and copper B, 353, 357, 393). The proPO has thiol ester-like motif (GCGWPQHV) which showed similar structural features of proPOs from other decapod crustaceans. It also contains three possible glycosylation sites (NES, NNT, NVN), and a conserved C-terminal region common to all known proPOs. Sequence comparison showed that the proPO deduced amino acid of lobster P. lognipes has an overall similarity of 75%, 69~72%, 67~68%, 65~68%, 60~68%, and 49~53% to that of lobster Homarus, penaeid shrimps, crab, freshwater prawn, crayfish, insects, respectively. The proPO was strongly expressed in hemocytes, but not in hepatopancreas, intestine, muscle, gill, and eyestalk. The proPO transcript of longlegged spiny lobster P. longipes increased significantly in 120 h post-glucan injection.

關鍵字

Panulirus longipes ； Prophenoloxidase ； β-glucan ； RT-PCR

並列摘要

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。

並列關鍵字

長足龍蝦 ； 原酚氧化酵素 ； 葡聚糖 ； 反轉錄-酵素聚合酶反應