Transcripts of developmentally regulated Plasmodium falciparum genes quantified by real-time RT-PCR
Transcripts of developmentally regulated Plasmodium falciparum genes quantified by real-time RT-PCR
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DOI：
10.1093/nar/30.10.2224
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发表时间：
2002-05-15
影响因子：
14.9
通讯作者：
Adams, JH
中科院分区：
文献类型：
--
作者：
Blair, PL;Witney, A;Adams, JH
恶性疟原虫红细胞内发育是一个复杂的过程。从营养获取和生长的滋养体阶段迅速发展到合成和生殖分裂体阶段，该阶段以产生新的侵入性分裂体结束。在这一过程中，疟原虫必须根据自身的代谢和合成需要表达一系列不同的基因产物。我们对分裂体后期形成的顶复合体的分殖子细胞器的发育特别感兴趣。我们首次使用实时荧光定量RT-PCR法（TaqMan(R)）对疟原虫进行了红细胞期特异性基因表达分析。我们分析了恶性疟原虫eba-175和其他红细胞结合样（ebl）家族基因在温度同步寄生虫中的转录本，发现ebl基因具有严格控制的阶段特异性转录。正如预期的那样，eba-175转录本仅在分裂体发育的末期丰富，其模式在ebl中最常见，包括baebl， pebbl和jesebl。maebl转录模式是独特的，在滋养体中后期达到高峰，但在分裂体后期没有。Ebl-1表现出另一种表达模式，在分裂中期达到峰值，然后在分裂晚期显著减少。我们的分析表明，使用实时荧光RT-PCR检测5'核酸酶是一种灵敏、定量的分析疟原虫转录本的方法。
Plasmodium falciparum intraerythrocytic development is a complex process. Development proceeds rapidly from the trophozoite phase of nutrient acquisition and growth through to the synthetic and reproductive schizont phase, which ends with production of new invasive merozoites. During this process, the malaria parasite must express a series of different gene products, depending on its metabolic and synthetic needs. We are particularly interested in the development of the merozoite's organelles in the apical complex, which form during the later schizont stages. We have used quantitative real-time RT-PCR fluorogenic 5' nuclease assays (TaqMan(R)) for the first time on malaria parasites for analysis of erythrocytic stage-specific gene expression. We analyzed transcripts of the P.falciparum eba-175 and other erythrocyte binding-like (eb l) family genes in temperature-synchronized parasites and found ebl genes have tightly controlled, stage-specific transcription. As expected, eba-175 transcripts were abundant only at the end of schizont development in a pattern most common among ebl, including baebl, pebl and jesebl. The maebl transcript pattern was unique, peaking at mid-late trophozoite stage, but absent in late-stage schizonts. ebl-1 demonstrated another pattern of expression, which peaked during mid-schizont stage and then significantly diminished in late-stage schizonts. Our analysis demonstrates that using real-time RT-PCR fluorogenic 5' nuclease assays is a sensitive, quantitative method for analysis of Plasmodium transcripts.