Ai Hua-Shui, Huang Yong-Chun, Li Se-Dong, Weng Shao-Ping, Yu Xiao-Qiang, He Jian-Guo
State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen (Zhongshan) University, 135 Xingang Road West, Guangzhou 510275, PR China.
Fish Shellfish Immunol. 2008 Jul;25(1-2):28-39. doi: 10.1016/j.fsi.2007.12.002. Epub 2008 May 12.
Previously, a prophenoloxidase (proPO) gene (named proPO-a here) from hemocytes of Litopenaeus vannamei was isolated. Here, a proPO-b gene was also identified and characterized from hemocytes of L. vannamei. The cDNA sequences of proPO-a and proPO-b were compared, and it was found that both proPOs had a microsatellite DNA site near the 3' end of the open reading frame (ORF). However, the microsatellite DNA of proPO-b contained a compound imperfect simple sequence repeats (SSR) ((CT)(38)(CA)(8)(AA)(CA)(3)(TA)(CA)(14)), which was different from the perfect one ((CT)(20)) of proPO-a, and the cDNA sequences of proPO-a and proPO-b prior to the microsatellite DNA were almost identical, but differed after the microsatellite DNA. ProPO-b (3232 bp) was longer than proPO-a (2471 bp). The 3' UTR sequence after SSR of proPO-a was not detected in shrimp randomly collected from five different geographically separate populations by reverse-transcription polymerase chain reaction (RT-PCR). On the contrary, the 3' UTR sequence of proPO-b was detected in all five groups of shrimps. Northern blot analysis showed that a transcript at approximately 3.2kb, but not 2.5kb, was detected mainly in hemocytes, and also present in midgut, gill, heart, stomach, posterior midgut cecum, and cuticular epidermis, but no signal was detected in hepatopancreas and musculature. RT-PCR and quantitative real-time RT-PCR analysis showed similar results of the proPO-b expression profile in these shrimp tissues. We also observed that proPO-b expression was down-regulated in shrimp challenged with white spot syndrome virus (WSSV). Our results suggest that proPO-b is a main transcript form of proPO gene in L. vannamei, and it may play a role in defence against WSSV virus.
先前，从凡纳滨对虾血细胞中分离出一种酚氧化酶原（proPO）基因（此处命名为proPO-a）。在此，还从凡纳滨对虾血细胞中鉴定并表征了一个proPO-b基因。比较了proPO-a和proPO-b的cDNA序列，发现这两种proPO在开放阅读框（ORF）的3'端附近都有一个微卫星DNA位点。然而，proPO-b的微卫星DNA包含一个复合不完全简单序列重复（SSR）（(CT)(38)(CA)(8)(AA)(CA)(3)(TA)(CA)(14)），这与proPO-a的完美微卫星DNA（(CT)(20)）不同，并且在微卫星DNA之前的proPO-a和proPO-b的cDNA序列几乎相同，但在微卫星DNA之后有所不同。proPO-b（3232 bp）比proPO-a（2471 bp）长。通过逆转录聚合酶链反应（RT-PCR）在从五个地理上不同的种群随机采集的对虾中未检测到proPO-a的SSR后的3'UTR序列。相反，在所有五组对虾中都检测到了proPO-b的3'UTR序列。Northern印迹分析表明，在大约3.2kb处检测到一个转录本，但在2.5kb处未检测到，该转录本主要在血细胞中检测到，也存在于中肠、鳃、心脏、胃、后中肠盲囊和表皮中，但在肝胰腺和肌肉组织中未检测到信号。RT-PCR和定量实时RT-PCR分析显示了这些对虾组织中proPO-b表达谱的相似结果。我们还观察到，在感染白斑综合征病毒（WSSV）的对虾中proPO-b表达下调。我们的结果表明，proPO-b是凡纳滨对虾中proPO基因的主要转录形式，并且它可能在抵御WSSV病毒中发挥作用。
