马尔尼菲青霉Rho GTPase激活因子RNA干扰载体的构建及其功能初步研究

发布时间：2019-07-04 10:41

【摘要】： 马尔尼菲青霉菌(Penicillium marneffei)是目前已知300余种青霉菌中唯一具有温度敏感的两相致病菌。它广泛分布于中国南部及东南亚地区,能引起呼吸道、皮肤以及系统性霉菌病,特别是在免疫功能低下和艾滋病病毒感染的患者中。随着艾滋病在全世界范围的蔓延,马尔尼菲青霉菌引起的急性系统性或播散性感染明显增加,已成为影响未来公众健康的潜在条件致病真菌。为更好的探讨马尔尼菲青霉菌的致病机制,识别其潜在的抗菌靶位,有必要对马尔尼菲青霉菌中一些与致病相关的重要基因进行深入的功能研究。然而,传统的基因功能研究方法低效且繁琐,给在真菌中开展大规模的基因功能研究带来了困难。近来,RNAi技术作为特异、高效、简便地基因功能研究方法给这些问题的解决带来了希望。本研究首次尝试了在马尔尼菲青霉菌中建立基于RNAi技术的基因功能研究平台。 Rho GTP酶(Rho GTPase)可能在控制细胞极性生长发育方面有着重要的作用,因而成为本研究的关注重点,即构建针对Rho GTPase上游激活因子的干扰载体,通过沉默Rho GTPase激活因子来初步探讨马尔尼菲青霉菌中RhoGTPase的功能。本研究构建以xylp为启动子,靶基因的正反双向序列被550bpGUS序列隔开的发夹结构载体PCB309A-XRGT,沉默靶基因。利用根癌农杆菌介导的方法转化马尔尼菲青霉,以博莱霉素为抗性标记进行突变子的筛选。结果表明,RhoGTPase激活因子的基因干扰后马尔尼菲青霉菌丝生长受到抑制,菌落明显变小,这与野生型相比发生显著变化。RT-PCR也检测到该基因mRNA水平的降低。鉴于Rho GTPase在生物中的重要作用和转录基因沉默体系得建立,我们对其基因功能进行了初步的探讨。推测该基因在马尔尼菲青霉菌菌落的形态形成过程有着重要的调控作用。这是首次成功应用RNAi技术,利用根癌农杆菌介导的转化方法研究马尔尼菲青霉中的重要基因功能,为深入研究Rho GTPase在马尔尼菲青霉菌的致病机制中所扮演角色提供技术手段,同时也为大规模地研究马尔尼菲青霉菌基因功能奠定了基础。
[Abstract]:Penicillium marneffei is the only temperature-sensitive two-phase pathogenic bacteria in more than 300 species of Penicillium. It is widely distributed in southern China and Southeast Asia, which can cause respiratory, skin and systemic mycosis, especially in patients with low immune function and HIV infection. With the spread of AIDS around the world, acute systemic or disseminated infections caused by Penicillium marneffei have significantly increased and have become a potential condition-causing fungus that has an impact on the future public health. In order to study the pathogenesis of Penicillium marneffei and identify its potential antibacterial target, it is necessary to carry out in-depth function of some important genes related to the pathogenesis of Penicillium marneffei. However, the traditional gene function research method is inefficient and tedious, and brings about a large-scale gene function research in the fungus. In recent years, RNAi technology has been used as a specific, efficient and simple gene function research method to solve these problems. Hope. This study first tried to establish a gene function based on RNAi technology in Penicillium marneffei. The Rho GTPase (Rho GTPase) may play an important role in controlling the growth and development of the cell. In this paper, the Rho GTPase activation factor is used to study the RhoGTPase in the mold of Penicillium marneffei. The function of se is that Xylp is used as a promoter, and the positive and negative two-way sequence of the target gene is separated from the 550 bpGUS sequence by the carrier PCB309A-XR. GT, silencing target gene. Transformed by Agrobacterium-mediated transformation of Penicillium marneffei with bleomycin as resistance marker The results showed that the growth of Penicillium marneffei was inhibited after the gene interference of the RhoGTPase activation factor, and the colony became smaller, which was similar to that of the wild type. The gene m was also detected by RT-PCR, as compared to the occurrence of a significant change. In view of the important role of Rho GTPase in the organism and the establishment of the silencing system of the transcription gene, we have an interest in the reduction of the RNA level. A preliminary study of the function of the gene is carried out. It is assumed that the gene is formed in the form of the colony of Penicillium marneffei. This is the first successful application of RNAi to study the important gene function in Penicillium marneffei by Agrobacterium-mediated transformation. In order to study the pathogenesis of Rho GTPase in Penicillium marneffei The role of the mechanism provides technical means, and is also a large-scale study of the Mal.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R379

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