红内期恶性疟原虫3D7中等长度ncRNA（RUF6-15）的功能研究

发布时间：2018-01-24 23:39

本文关键词： 疟疾恶性疟原虫 RUF6-15 Var　出处：《北京协和医学院》2016年博士论文　论文类型：学位论文

【摘要】：疟疾是一种十分严重的热带病,在感染人的4中疟原虫中恶性疟原虫的毒性最强,死亡率极高。疟原虫的生活史复杂,需要两种类型的宿主-雌性按蚊和人。在人体内有红外期和红内期两个发育阶段,但是其主要临床症状发生在红内期阶段。全基因组测序的完成及后继的芯片技术、高通量测序技术的快速发展,恶性疟原虫中的非编码RNA (ncRNA)也越来越引起人们的关注。ncRNA从长度上可以分为3类：小于50 nt的小片段ncRNA; 50 nt到500 nt的中等长度ncRNA;大于500 nt的长链ncRNA (Long non-coding RNA, lncRNA)。通过数据库数据比较基因组学分析,在恶性疟原虫中没有找到Dicer、Piwi、PAZ或RdRp的同源区域,因此认为恶性疟原虫中没有内源性小RNAs的存在。因此,中等长度的ncRNA引起了广泛关注。对于疟原虫中等长度组成型ncRNA的研究比较成熟,如tRNA、rRNA这些RNA不被翻译成蛋白质,但是参与蛋白质的翻译过程：此外还有snRNA、 snoRNA等参与RNA剪接和RNA修饰。Chakrabarti等鉴定出6个新的未知功能的RNA (RNAs of unknown function,简称RUFs),其中RUF5、6仅在恶性疟原虫与里氏疟原虫中存在,RUF6包括15条高度同源的序列,长度在144-160 nt,这些同源序列位于RIFIN或VAR的基因间区。恶性疟原虫变异抗原基因(Var基因)家族编码的恶性疟原虫红细胞膜蛋白1(PfEMP1)是介导恶性疟原虫抗原变异和红细胞粘附微血管的媒介。PfEMP1由恶性疟原虫分泌产生,然后转移到感染的红细胞膜上,并在红细胞表面蓄积而形成结节,PfEMP1介导的细胞粘附和恶性疟原虫的致病性密切相关。这些RUFs是否参与了Var基因的调控以及调节疟原虫红细胞内期的发育分化及其内在调节机制仍不清楚。本研究通过Northern blot、FISH、转染、RNA-Seq、qRT-PCR、双荧光素酶实验等实验首次揭示了RUF6中第15条同源簇(RUF6-15)的生物学功能。在野生型3D7虫株每一个生活周期中RUF6-15中的转录水平是随着生长发育时间的延长越来越多,转录后位于细胞核内参与调控作用。转染过表达RUF6-15的虫株与对照株相比生长速度更快,敲低RUF6-15的虫株与对照株相比生长速度变慢。通过过表达RUF6-15后转录本水平的高通量测序结果及实验室验证结果显示,RUF6-15的靶基因为var基因家族。进一步通过双荧光素酶实验验证了RUF6-15是通过结合Var基因5’UTR区来调控var的表达。粘附实验表明过表达RUF6-15的虫株粘附能力有明显的提升,RUF6-15敲低的虫株粘附能力有所降低。本研究首次揭示了RUF6-15的生物学功能及作用机制,对深入认识疟疾的发病机制和制定新的防治措施均意义重大。
[Abstract]:Malaria is a very serious tropical disease. The virulence and mortality of Plasmodium falciparum are the highest among the 4 infected people. The life history of Plasmodium falciparum is complex. Two types of hosts are needed-female Anopheles and humans. There are two stages of development in the human body: infrared and red. However, the main clinical symptoms occurred in the red phase. Complete genome sequencing and subsequent chip technology, high-throughput sequencing technology rapid development. The non-coding RNA ncRNAs in Plasmodium falciparum have attracted more and more attention. The length of ncRNAs can be divided into three categories: small fragments of ncRNAs less than 50 NT; Medium length ncRNAs from 50 NT to 500 NT; Long strand ncRNA long non-coding, LNC RNAs > 500nt were used to compare genomics analysis with database data. There is no homologous region of Dicerus Piwipas PAZ or RdRp in Plasmodium falciparum, so it is believed that there is no endogenous small RNAs in Plasmodium falciparum. Medium-length ncRNA has attracted much attention. The studies on the medium-length ncRNA of Plasmodium malaria are more mature, such as tRNA-rRNA, which are not translated into proteins. But involved in the protein translation process: there is also snRNA. SnoRNA et al. participated in RNA splicing and RNA modification. Chakrabarti et al. identified six new unknown functions of RNA (. RNAs of unknown function. Among them, RUF6 contains only 15 highly homologous sequences in Plasmodium falciparum and Plasmodium Rei, with a length of 144-160 NT. These homologous sequences are located in the intergenic region of RIFIN or VAR. Plasmodium falciparum variant antigen gene (Var) family encodes erythrocyte membrane protein 1 of Plasmodium falciparum PfEMP1). PfEMP1 is a vector that mediates the variation of Plasmodium falciparum antigen and the adhesion of erythrocytes to microvessels. PfEMP1 is secreted by Plasmodium falciparum. It then metastasizes to the infected erythrocyte membrane and accumulates on the surface of the erythrocyte to form nodules. The cell adhesion mediated by PfEMP1 is closely related to the pathogenicity of Plasmodium falciparum. Do these RUFs participate in the regulation of Var gene and regulate the development and differentiation of the erythrocyte phase of Plasmodium falciparum and its intrinsic regulation? The mechanism is still unclear. This study is based on Northern. Blot. Fish, transfection of RNA-Seqsil-qRT-PCR. Double luciferase experiments revealed for the first time the 15th homology cluster in RUF6, RUF6-15). The transcriptional level of RUF6-15 in each life cycle of wild-type 3D7 strain is increasing with the prolongation of growth and development time. The post-transcriptional locus involved in the regulation of the cell nucleus. The growth rate of the transfected RUF6-15 strain was faster than that of the control strain. The growth rate of the strain with low RUF6-15 was slower than that of the control strain. The results of high throughput sequencing and laboratory verification showed that the expression of RUF6-15 post-transcripts level was higher than that of the control strain. The target gene of RUF6-15 is the var gene family. Further, the double luciferase experiment proved that RUF6-15 regulates the expression of var by binding to the 5UTR region of Var gene. The results showed that the adhesion ability of RUF6-15 overexpression strains was significantly improved. The adhesion ability of RUF6-15 knockout was decreased. This study revealed the biological function and mechanism of RUF6-15 for the first time. It is of great significance to understand the pathogenesis of malaria and to establish new control measures.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R382

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