须癣毛癣菌转录组测序分析及ZafA基因遗传转化用双元载体的构建

发布时间：2018-03-20 12:18

  本文选题：须癣毛癣菌　切入点：转录组测序　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：须癣毛癣菌(Trichophyton Mentagrophytes)是常见的人畜共患皮肤癣菌病病原,可引起严重的皮肤感染,但目前常用的真菌性皮肤病的防治手段相对简单,对于该菌的研究仍大都停留在流行病学和药物疗效等方面,已不能满足临床医学的需求。在真菌等微生物的生长和繁殖中,锌是一种重要的微量营养元素,其常与多种功能蛋白结合以形成锌指蛋白来完成生物学功能。目前,多种真菌的锌吸收转运调控因子已经明确,如:烟曲霉的ZafA蛋白、酵母菌的Zap1蛋白等,此类蛋白作为锌响应活力因子可在转录水平上对多种锌转运蛋白的表达进行调控,从而控制真菌对锌离子的摄取,在真菌的生长和致病过程中扮演重要角色。但目前,须癣毛癣菌的锌响应活力因子的基因组成及功能还未确定,锌吸收转运调控网络尚不清楚,确定须癣毛癣菌中锌响应活力因子以及锌转运蛋白的基因组成对于须癣毛癣菌致病性的研究具有重要意义。研究发现多种真菌的锌响应活力因子的基因组成具有一定的相似性,初期试验,以烟曲霉锌响应活力因子的基因序列为基础设计引物,以须癣毛癣菌全基因组DNA、cDNA为模板进行PCR扩增,经过多次试验发现无法获得须癣毛癣菌锌响应活力因子的基因组成。因此,我们使用Illumina HiSeqTM 2000技术对正常状态下及锌缺乏情况下的须癣毛癣菌进行了转录组测序,并对测序数据进行了从头组装、基因功能注释以及基因的差异表达分析等,后续通过BLASTX序列相似性搜索以及对须癣毛癣菌转录组测序数据的分析确定出须癣毛癣菌锌响应活力因子的基因组成,初步推断出与锌吸收转运相关的其他基因;并使用重组克隆技术构建了针对ZafA基因的遗传转化用双元载体,为后续的基因功能验证奠定了基础。研究结果如下:1使用Illumina测序平台对须癣毛癣菌进行了RNA-seq,共获得10751个unigenes,89%以上的基因得到功能注释,对于须癣毛癣菌的基因功能研究以及基因组图谱的组装提供了宝贵的数据支持。2通过与烟曲霉ZafA基因、酿酒酵母Zap1基因进行序列同源性比对以及功能注释分析等,确定了须癣毛癣菌中与锌吸收转运相关的基因并高保真扩增出须癣毛癣菌ZafA基因。3将ZafA基因左右侧翼片段以及来自质粒pAN7-1的潮霉素B抗性基因(hph)同时插入到双元载体pDHt/SK中,构建ZafA基因转化用双元载体pDHt/ZafAⅠ、Ⅱ::hph,经PCR及酶切验证,双元载体构建成功。试验结果表明,本研究完成了须癣毛癣菌的转录组测序及功能注释;确定了须癣毛癣菌ZafA基因的组成,并成功地构建了转化用双元载体pDHt/ZafAⅠ、Ⅱ::hph,为之后的ZafA基因的毒力试验及功能验证奠定了基础。
[Abstract]:Trichophyton Mentagrophytes (Trichophyton Mentagrophytes) is a common pathogen of zoonotic dermatophytosis, which can cause severe skin infection, but the prevention and treatment of fungal dermatosis is relatively simple. Most of the studies on this bacterium are still focused on epidemiology and drug efficacy. Zinc is an important micronutrient element in the growth and reproduction of fungi and other microbes. It often binds with a variety of functional proteins to form zinc finger proteins to complete biological functions. At present, zinc absorption and transport regulators of many fungi, such as ZafA protein of Aspergillus fumigatus and Zap1 protein of yeast, have been identified. These proteins can regulate the expression of many zinc transporters at the transcriptional level, thus controlling the uptake of zinc ions by fungi and playing an important role in the growth and pathogenicity of fungi. The gene composition and function of zinc responsive activity factor of Trichophyton rubrum were not determined, and the regulatory network of zinc absorption and transport was not clear. It is important to determine the zinc responsive active factor and zinc transporter gene composition in Trichophyton tubuloides for the study of pathogenicity of Trichophyton tinea. It is found that the gene composition of zinc responsive activity factor of many fungi has certain similarity. In the initial experiment, primer was designed based on the gene sequence of zinc response activity factor of Aspergillus fumigatus, and the whole genome DNA of Trichophyton tuber was used as template for PCR amplification. After many experiments, it was found that the gene composition of zinc response activity factor of Trichophyton tubuloides could not be obtained. Therefore, we used Illumina HiSeqTM 2000 technique to sequence the transcriptome of Trichophyton rubrum in normal condition and zinc deficiency. The sequencing data were assembled from scratch, gene function annotation and gene differential expression analysis, etc. The BLASTX sequence similarity search and the analysis of the transcriptome sequencing data of Trichophyton versicolor confirmed the gene composition of zinc response activity factor of Trichophyton mutans and preliminarily inferred other genes related to zinc absorption and transport. Using the recombinant cloning technique, a binary vector for genetic transformation of ZafA gene was constructed. The results are as follows: 1 used Illumina sequencing platform to carry out RNA-seq. a total of 10 751 genes of Unigenesus were annotated. It provides valuable data for the study of the gene function of Trichophyton tinea and the assembly of genome map. 2. Through sequence homology alignment and functional annotation analysis with ZafA gene of Aspergillus fumigatus, Zap1 gene of Saccharomyces cerevisiae, etc. The genes related to zinc absorption and transport in Trichophyton tinea were identified and the ZafA gene of Trichophyton trichoriasis was amplified by high fidelity amplification. The ZafA gene fragment from left and right flanks and the hygromycin B resistance gene from plasmid pAN7-1 were inserted into the binary vector pDHt/SK at the same time. The bivariate vectors pDHt/ZafA 鈪，

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