Division of Fisheries Biotechnology & Molecular Biology, Department of Biotechnology, Faculty of Science and Humanities, SRM University, Kattankulathur 603 203, Chennai, Tamil Nadu, India.
Gene. 2013 Jul 25;524(2):139-51. doi: 10.1016/j.gene.2013.04.044. Epub 2013 Apr 29.
The copper containing prophenoloxidase enzyme plays a crucial role in the defense system of arthropods, especially crustaceans and insects. In this study, we have reported a full length cDNA of prophenoloxidase identified from the constructed cDNA library of freshwater prawn Macrobrachium rosenbergii by genome sequence FLX technology. The identified full length M. rosenbergii prophenoloxidase (MrProPO) consists of 3378 base pairs (bp) with an open reading frame (ORF) of 2099 bp. This ORF encoded a polypeptide of 700 amino acids (aa) with an estimated molecular mass of 80 kDa and a predicted isoelectric point (pI) of 6.7. The motif analysis of MrProPO shows two copper binding sites (CuA and CuB) along with hemocyanin signatures and a thiol-ester like motif. MrProPO exhibited the maximum similarity (97%) with ProPO from Macrobrachium nipponense and is closely clustered with other crustacean ProPO in the phylogenetic tree. Bioinformatics analysis suggests that MrProPO is a member of the prophenoloxidase family, due to the conserved domains, motifs and similarity with other known ProPOs. The 3D structural analysis of MrProPO reveals that it has more random coils, moderate α-helices, few extended β-sheets and a very few β-turns. Among the 700 aa of MrProPO, 355 (50.71%), 206 (29.43%), 110 (15.71%) and 29 (4.14%) amino acids are responsible for random coils, α-helices, extended β-sheets and β-turns respectively. The gene expression results indicate MrProPO is widely distributed in all the tissues studied, but significantly (P<0.05) highest expression was observed in hepatopancreas. The relative expression of mRNA was quantified in hepatopancreas after being infected with virus [white spot syndrome baculovirus (WSBV) and M. rosenbergii nodovirus (MrNV)] and bacteria (Aeromonas hydrophila and Vibrio harveyi) using real-time PCR. MrProPO mRNA transcription significantly (P<0.05) increased at 24h post injection (p.i.) with subsequent decrease at 48 h p.i. in both viral and bacterial infected prawns. The highest enzyme activity was observed in hepatopancreas, which was also significantly higher (P<0.05) than detected in other tissues. Similar to gene expression results, the enzyme activity reached the peak at 24h p.i. and then the activity started decreasing. Overall results indicate that MrProPO is very likely to participate in the acute response against pathogen entry in prawns.
铜结合原酚氧化酶在节肢动物（尤其是甲壳类和昆虫）的防御系统中起着至关重要的作用。在本研究中，我们通过基因组序列 FLX 技术从淡水虾 Macrobrachium rosenbergii 构建的 cDNA 文库中报道了全长原酚氧化酶 cDNA 的鉴定。鉴定的全长 M. rosenbergii 原酚氧化酶（MrProPO）由 3378 个碱基对（bp）组成，开放阅读框（ORF）为 2099bp。该 ORF 编码一个 700 个氨基酸（aa）的多肽，估计分子量为 80kDa，预测等电点（pI）为 6.7。MrProPO 的基序分析显示，它有两个铜结合位点（CuA 和 CuB），以及血蓝蛋白特征和硫酯样基序。MrProPO 与 Macrobrachium nipponense 的 ProPO 具有最大的相似性（97%），并在系统发育树中与其他甲壳类 ProPO 密切聚类。生物信息学分析表明，MrProPO 是原酚氧化酶家族的成员，这是由于其保守结构域、基序和与其他已知 ProPO 的相似性。MrProPO 的 3D 结构分析表明，它具有更多的无规卷曲、适度的α-螺旋、较少的扩展β-折叠和非常少的β-转角。在 MrProPO 的 700 个氨基酸中，355（50.71%）、206（29.43%）、110（15.71%）和 29（4.14%）个氨基酸分别负责无规卷曲、α-螺旋、扩展β-折叠和β-转角。基因表达结果表明，MrProPO 在所有研究的组织中均有广泛分布，但在肝胰腺中表达水平显著（P<0.05）最高。通过实时 PCR 定量分析了感染病毒[白斑综合征杆状病毒（WSBV）和 M. rosenbergii 诺达病毒（MrNV）]和细菌（嗜水气单胞菌和哈维弧菌）后肝胰腺中 MrProPO mRNA 的相对表达。MrProPO mRNA 转录在病毒和细菌感染虾后 24 小时注射（p.i.）时显著（P<0.05）增加，随后在 48 小时 p.i.时减少。在肝胰腺中观察到最高的酶活性，也明显高于其他组织（P<0.05）。与基因表达结果相似，酶活性在 24 小时 p.i.时达到峰值，然后开始下降。总体结果表明，MrProPO 很可能参与了虾对病原体入侵的急性反应。
