红内期恶性疟原虫is-ncRNAs的功能研究

Malaria remains one of three most severe human infectious diseases worldwide, and the unicellular eukaryote, Plasmodium falciparum (P. falciparum), is the etiological agent of the most virulent and lethal form of malaria in human. The expression of the pathogeny related genes is variable. However, little is known about the gene expression regulation mechanisms of the parasite. Existing studies suggest that microRNAs, which are small regulatory ncRNAs that have been identified and characterized as vital components in most eukaryotic organisms, have not yet been discovered in P. falciparum by traditional cloning and sequencing methods. During the preliminary work in our lab, ncRNAs ranging from 50-50 nt in lenth, referred as intermediate-size ncRNA (is-ncRNA), of the intraerythrocytic P. falciparum had been identified and analyzed systematically. A total of 1,198 novel is-ncRNA candidates were obtained. In this study, P. falciparum strains with is-ncRNA(s) knocked out will be selected using the piggyBac transposon system and the function of particular is-ncRNAs will be studied. Target genes of the is-ncRNAs will be identified by comparing the differences between the is-ncRNA knocked out and normal P. falciparum strains in both the transcriptomics and proteomics. Functionalizing the is-ncRNA space will elucidate the regulatory roles of the is-ncRNAs in development of the intraerythrocytic P. falciparum. This study will undoubtedly help better understanding of the gene expression regulation mechanisms of the parasite and will lead to important insight about development of the antimalarial drugs and malaria vaccines.

疟疾是严重危害人类健康的三大传染病之一，恶性疟原虫是导致人类疟疾感染毒力最强、致死率最高的疟原虫虫种。疟原虫病原相关基因表达多变，而目前人们对其基因表达调控机制的认识还很匮乏。疟原虫缺乏内源性microRNA这一广泛存在于真核生物中的调节性小非编码RNA，提示有其它类型ncRNA发挥关键调控作用。本课题前期工作系统地分析和鉴定了红内期恶性疟原虫长度在50-500nt之间的ncRNA(is-ncRNA)，共获得1,198条新is-ncRNA。本研究将利用piggyBac转座子系统筛选is-ncRNA敲除虫株，进一步研究特定is-ncRNA的功能。通过比较敲除虫株和正常虫株在转录组和蛋白质组方面的差异鉴定is-ncRNA的靶基因，明确is-ncRNA对红内期恶性疟原虫基因表达的调控作用。该研究将加深人们对疟原虫基因表达调控机制的了解，为抗疟药靶点和疟疾疫苗的研发提供新思路。

疟疾是严重危害人类健康的三大传染病之一，恶性疟原虫（Plasmodium falciparum，P. falciparum）是导致人类疟疾感染毒力最强、致死率最高的疟原虫虫种。恶性疟原虫发育中经历多种形态学变化，其抗原蛋白具有相互排斥表达特点，这些生物学特点势必与其具有精密的基因表达调控机制有关。然而人们对其表达调控机制的认识一直比较有限。近些年来研究者们发现恶性疟原虫中具有丰富的非编码RNA（Non-coding RNA, ncRNA），其中一部分ncRNA 已被证实与恶性疟原虫生长发育和致病过程有关。特别是一些长链非编码RNA（Long Non-coding RNA, lncRNA）已被证实与恶性疟原虫var基因的表达调控有关。然而受研究方法和研究目的的影响，目前关于红内期恶性疟原虫lncRNA的表达谱还缺乏全面的认识；另外，目前人们并未找到恶性疟原虫内源性microRNA，因此对其lncRNA的功能也值得深入研究。本研究采用链特异性lncRNA 文库构建和高通量Illumina/Solexa双末端测序技术相结合的策略对红内期P. falciparum 3D7 的lncRNA表达谱进行了研究。后续针对形态差异最大的环期和裂殖体期的lncRNA进行了详细的生物信息学分析，结果我们发现了31条在新的lncRNAs，并对它们进行了实验验证。进一步我们利用RACE技术对其中的lncRNA30进行了全长鉴定；利用CRISPR/Cas9技术分别构建了lncRNA30过表达和低表达虫株，并对lncRNA30的功能展开研究。现有结果表明，与lncRNA30过表达后可促进恶性疟原虫生长，并会信号识别颗粒蛋白分子具有相互作用。详细机制研究有待深入探讨。本研究将加深人们对疟原虫基因表达调控机制的认识，从而为抗疟药物靶点的选择提供必要的理论依据。