Identification, characterization of functional candidate genes for host-parasite interactions in entomopathogenetic nematode Steinernema carpocapsae by suppressive subtractive hybridization
Identification, characterization of functional candidate genes for host-parasite interactions in entomopathogenetic nematode Steinernema carpocapsae by suppressive subtractive hybridization
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DOI：
10.1007/s00436-008-1030-4
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发表时间：
2008-08-01
影响因子：
2
通讯作者：
Simoes, Nelson
中科院分区：
文献类型：
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作者：
Hao, You-Jin;Montiel, Rafael;Simoes, Nelson
鉴定线虫分泌蛋白的寄生基因是了解线虫寄生昆虫分子基础的关键。本文通过筛选大蜡螟（Galleria mellonella）血淋巴诱导的线虫（Steinernema carpocapsae）cDNA消减文库，发现了一个含有2个内含子和3个外显子的编码半胱氨酸蛋白酶抑制剂的cDNA。全长cDNA包含一个开放阅读框，编码一个139个氨基酸的蛋白质，命名为Sc-cys，具有19个残基的信号肽。预测成熟蛋白质具有12，531.59 Da的分子量，9.44的pI，一个二硫键和三个保守结构域，据信对于抑制半胱氨酸蛋白酶是重要的。在基本局部比对和搜索工具分析中，推定的蛋白质前体与大量半胱氨酸蛋白酶抑制剂或半胱氨酸蛋白酶抑制剂样蛋白质显示出26-42%的同一性。系统进化分析表明，该蛋白可能是cystatin家族2的新成员.反转录北方印迹、半定量逆转录聚合酶链反应（RT-PCR）和实时荧光定量RT-PCR分析表明，昆虫血淋巴诱导后Sc-cys的表达水平显著上调。对分泌蛋白基因的特异性分析提供了一个可能的寄生虫基因表达谱。carpocapsae在整个寄生周期。
Identifying parasitism genes encoding proteins secreted from nematodes is the key to understanding the molecular basis of nematode parasitism to insects. In this paper, a cDNA with two introns and three exons encoding a cysteine protease inhibitor was identified by screening a cDNA subtractive library constructed from the nematode, Steinernema carpocapsae, induced by Galleria mellonella hemolymph. The full-length cDNA contains an open reading frame encoding a 139-amino acid protein, designated Sc-cys, with a 19-residue signal peptide. The mature protein was predicted to have a molecular weight of 12,531.59 Da, a pI of 9.44, one disulfide bond, and three conserved domains believed to be important for the inhibition of cysteine proteases. In Basic Local Alignment and Search Tool analyses, the putative protein precursor displayed 26-42% identities to a multitude of cystatins or cystatin-like proteins. Phylogenetic analysis suggested the novel cystatin is likely a new member of the family 2 cystatins. Reverse northern blot, semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and real-time RT-PCR analyses showed that the expression level of Sc-cys was upregulated substantially after induction by insect hemolymph. The specific analysis of genes encoding secretory proteins is providing a profile of putative parasitism genes expressed in S. carpocapsae throughout the parasitic cycle.