应用RNA干扰技术对新生隐球菌CNLAC1基因功能的初步研究

发布时间：2018-07-10 10:37

本文选题：新生隐球菌 + RNA干扰　；参考：《第二军医大学》2005年硕士论文

【摘要】：研究背景: 隐球菌病是新生隐球菌感染所致的全身感染性疾病,好发于细胞免疫功能低下患者。近年来隐球菌感染发生率呈明显上升的趋势,突出表现在爱滋病人群中,成为AIDS患者最常见的并发症之一,也是AIDS患者死亡的首要原因。 2000年3月启动的新生隐球菌基因组计划由斯坦福大学基因组技术中心(SGTC)和美国国家基因组中心(TIGR)联合于近期完成,为我们下一步的研究奠定了坚实的基础。但破解基因组的序列只是一个起点,许多毒性相关基因的确切致病机理仍有待研究。要对某一基因的功能进行研究,按照经典的分子遗传学方法,就必须应用同源重组的方法将该基因敲除,得到人工突变株,在此平台基础上,我们才可以通过该转化子的形态、结构、功能、传代过程和生理生化表现及其毒力的改变深入地研究该基因的功能。不过应用基因同源重组进行相关基因敲除来研究其功能,耗时费力且成功率低,为全世界科研工作者所遇到的共同难题。而近年来兴起的RNA干扰技术(RNAi),即用20多个核苷酸组成的短的双链RNA(siRNA)进行转录后基因沉默,已经迅速而广泛地应用到基因功能,基因表达调控机制研究等热门领域,·RNA干扰技术作为一种简单、有效的代替基因敲除的遗传工具,正在功能基因组学领域掀起一场真正的革命,并将彻底改变这个领域的研究步伐,即如果在实验设计中,在靶细胞中引入长双链RNA中有与靶基因所转录的mRNA同源的序列,即可触发“扳机”,引起特定的转录后基因沉默,使该基因功能丧失或部分丧失,从而明确其功能。研究目的: (1) 构建RNA干扰质粒Pcnlacl-tel,正义RNA质粒Pcnlacl-S-tel,反义RNA质粒Pcnlacl-A-tel的构建 (2) 摸索隐球菌转化的条件,进行一整套转化体系的构建 (3) 应用RNA干扰技术研究酵母的基因功能,建立了一套完整的酵母基因功能研究体系及技术平台,进一步推广至其它酵母的基因功能研究。
[Abstract]:Background: Cryptococcosis is a systemic infectious disease caused by Cryptococcus neoformans infection. In recent years, the incidence of Cryptococcus infection is on the rise, especially in the AIDS population, becoming one of the most common complications of AIDS patients. The Cryptococcus neoformans Genome Project, launched in March 2000, was launched by the Stanford University Center for Genome Technology (SGTC) and the United States. The National Genome Center (TIGR) was jointly completed in the near future. For our next research laid a solid foundation. However, the sequence of genome is only a starting point, and the exact pathogenesis of many toxic genes remains to be studied. In order to study the function of a gene, according to the classical molecular genetic method, we must use homologous recombination to knock out the gene and get the artificial mutant. On the basis of this platform, we can pass the morphology of the transformant. The structure, function, passage, physiological and biochemical characteristics and virulence of the gene were studied in depth. However, using homologous recombination to study the function of gene knockout is time-consuming and low success rate, which is a common problem for researchers all over the world. In recent years, RNA interference (RNAi), which is composed of more than 20 nucleotides of short double-stranded RNA (siRNA) for post-transcriptional gene silencing, has been rapidly and widely used in gene function. RNA interference, as a simple and effective genetic tool instead of gene knockout, is leading a real revolution in the field of functional genomics. And will completely change the pace of research in this field, that is, if the target cells are designed to introduce a sequence homologous to the mRNA transcripted by the target gene in a long double-stranded RNA, the trigger can be triggered, causing a specific post-transcriptional gene silencing. Loss or partial loss of function of the gene, thereby clarifying its function. Objective: (1) to construct RNA interference plasmid Pcnlacl-tel, sense RNA plasmid Pcnlacl-S-teland antisense RNA plasmid Pcnlacl-A-tel. (2) explore the condition of Cryptococcus transformation, Construction of a complete set of transformation systems (3) RNA interference technique was used to study the gene function of yeast. A complete research system and technical platform of yeast gene function was established, which was further extended to other yeast gene function research.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R379.5

【共引文献】