口虾蛄proPO基因全长cDNA的克隆与组织表达  

Full length cDNA cloning and tissue expression of prophenoloxidase from Oratosquilla oratoria

作　　者：刘海映[1] 刘连为[1] 姜玉声[2] 隋宥珍[2] 陈雷[1] 

机构地区：[1]大连海洋大学辽宁省海洋牧场工程技术研究中心,大连116023 [2]大连海洋大学农业部海洋水产增养殖学与生物技术重点开放实验室,大连116023

出　　处：《生态学报》2013年第6期1713-1720,共8页Acta Ecologica Sinica

基　　金：国家海洋公益性行业科研专项经费项目(200905019);辽宁省科学技术计划项目(2008203002);辽宁省自然科学基金项目项(20052138)

摘　　要：口虾蛄是海湾底拖网渔业中具有重要经济价值的种类,分布广泛。其自然资源日渐衰退,人工育苗技术获得成功后,口虾蛄养殖过程中病害问题及其防治应引起足够的重视。为此,拟通过分子生物学手段研究口虾蛄免疫系统的核心酶——酚氧化酶(PO)的分子结构及该基因的组织表达,从而在分子水平上深入探究其免疫机理。采用反转录PCR(RT-PCR)与cDNA末端快速克隆(RACE)技术从口虾蛄血细胞中克隆了酚氧化酶原(O-proPO)基因,cDNA全长为2436bp,其中开放阅读框为2142bp,编码713个氨基酸。预测分子量为82446Da,等电点(pI)为8.78。该基因与Genbank上登录的斑节对虾、凡纳滨对虾、罗氏沼虾、短沟对虾、日本对虾proPO基因序列具有较高的同源性,分别为82%、76%、76%、72%、70%。序列分析表明O-proPO为proPO家族中的一个成员,其氨基酸序列中含有多个免疫调节作用位点。系统进化分析显示O-proPO与十足目种类的proPO为同一分支的2个亚群,而后于丰年虫的proPO形成一分支。O-proPO基因表达具有组织特异性,在血淋巴和肠中表达,但在血淋巴中表达量明显高于肠。Prophenoloxidase （proPO） is the terminal component of the proPO activating system, a defense and recognition system in crustaceans and insects. It can be activated to generate the active enzyme phenoloxidase （PO） by a proteinase cascade and some additional factors. PO can oxidize phenols to quinones that can then polymerize non-enzymatically to melanin. Recently, molecular biology studies of proPO, such as gene expression, structure analysis, and real time expression, have made great progress. However, these studies focused mainly on shrimps and crabs. To better understand the phylogenetic relations and characteristics of the proPOs gene among crustaceans, it is necessary to study crustaceans other than shrimp and crab. Oratosquilla oratoria belongs to order Stomatopoda in phylum Crustacea. O. oratoria is found on muddy bottoms in the coastal waters of Russia, China, Korea, Japan, Vietnam, and Australia and is exploited commercially in many regions. Its natural resources have degraded by degrees since fishing intensification increased and the ecological environment of inshore areas deteriorated. Artificial breeding of O. oratoria is now practiced successfully. However, just as the sustainability and development of shrimp aquaculture is increasingly threatened by significant ecological and pathological problems, it is likely that O. oratoria aquaculture will develop similar severe issues. To boost the healthy development of O. oratoria aquaculture, much more attention should be paid to the PO enzyme which plays an important part in the immune system of crustaceans. Research on the molecular structure and tissue expression of the proPO gene may give us a more thorough understanding of the immunologic mechanisms in O. oratoria. In this paper, the cDNA of the O. oratoria proPO （O-proPO） gene was cloned using reverse transcription PCR （RT-PCR） and rapid amplification of cDNA ends （RACE）. The full length cDNA of O-proPO consisted of 2, 436 base pairs （bp） with a 2, 142 bp open reading frame （O

关 键 词：口虾蛄 酚氧化酶原 分子克隆 表达分析 

分 类 号：Q78[生物学—分子生物学]