Yeh Maw-Sheng, Lai Ching-Yi, Liu Chun-Hung, Kuo Ching-Ming, Cheng Winton
Department of Food and Nutrition, Hungkuang University, Sha-Lu, Taichung 43302, Taiwan.
Fish Shellfish Immunol. 2009 Jan;26(1):49-55. doi: 10.1016/j.fsi.2008.10.003. Epub 2008 Oct 18.
Prophenoloxidase (proPO) is a melanin-synthesising enzyme that plays important roles in immune responses by crustaceans. Previously, we cloned and characterized proPO-I from white shrimp, Litopenaeus vannamei. In the present study, a novel prophenoloxidase-II (proPO-II) cDNA was also cloned from haemocytes of L. vannamei using oligonucleotide primers and reverse-transcriptase polymerase chain reaction (RT-PCR). Both 3'- and 5'-regions were isolated by the rapid amplification of complementary (c)DNA end (RACE) method. The 2504-bp cDNA contained an open reading frame (ORF) of 2073 bp, an 84-bp 5'-untranslated region, and a 347-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid sequence (691 amino acids) was 78.8 kDa with an estimated pI of 6.07. It contains two putative tyrosinase copper-binding motifs and a conserved C-terminal region common to all known proPOs. Comparisons of the amino acid sequences showed that white shrimp proPO-II is more closely related to the proPO of other penaeids than to that of crayfish, lobsters, crab, or a freshwater prawn, and is the ancestor type of known penaeid proPOs. proPO-I and proPO-II messenger (m)RNAs of shrimp were located on different loci, and were constitutively expressed mainly in haemocytes. The transcriptional regulation of these two proPOs in shrimp at different molt stages, those administered dietary sodium alginate, and those challenged with Vibrio alginolyticus were surveyed. The results showed that the proPOs may be directly involved in the acute-phase immune defence, and proPO-II may contribute earlier to immune defence in shrimp injected with V. alginolyticus, and it may be regulated by ecdysone. However, a similar effect was found by stimulating proPO-I and proPO-II mRNA expression in shrimp fed a sodium alginate-containing diet. Results of this study provide a basis for developing a comprehensive understanding of expression/function relationships of individual proPOs in shrimp.
酚氧化酶原（proPO）是一种合成黑色素的酶，在甲壳类动物的免疫反应中发挥重要作用。此前，我们从凡纳滨对虾（Litopenaeus vannamei）中克隆并鉴定了proPO-I。在本研究中，还使用寡核苷酸引物和逆转录聚合酶链反应（RT-PCR）从凡纳滨对虾的血细胞中克隆了一种新的酚氧化酶原-II（proPO-II）cDNA。通过互补（c）DNA末端快速扩增（RACE）方法分离出3'和5'区域。2504 bp的cDNA包含一个2073 bp的开放阅读框（ORF）、一个84 bp的5'非翻译区和一个347 bp的包含多聚A尾的3'非翻译区。推导的氨基酸序列（691个氨基酸）的分子量为78.8 kDa，估计pI为6.07。它包含两个假定的酪氨酸酶铜结合基序和所有已知proPO共有的保守C末端区域。氨基酸序列比较表明，凡纳滨对虾proPO-II与其他对虾科动物的proPO比与小龙虾、龙虾、螃蟹或淡水虾的proPO关系更密切，并且是已知对虾科动物proPO的祖先类型。对虾的proPO-I和proPO-II信使（m）RNA位于不同位点，并且主要在血细胞中组成性表达。研究了这两种proPO在不同蜕皮阶段、投喂海藻酸钠饲料以及受到溶藻弧菌攻击的对虾中的转录调控。结果表明，proPO可能直接参与急性期免疫防御，并且proPO-II可能在注射溶藻弧菌的对虾中更早地对免疫防御做出贡献，并且它可能受蜕皮激素调节。然而，在投喂含海藻酸钠饲料的对虾中刺激proPO-I和proPO-II mRNA表达也发现了类似的效果。本研究结果为全面了解对虾中单个proPO的表达/功能关系提供了基础。
