本文選題：須癬毛癬菌　切入點(diǎn)：轉(zhuǎn)錄組測(cè)序　出處：《西北農(nóng)林科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：須癬毛癬菌(Trichophyton Mentagrophytes)是常見的人畜共患皮膚癬菌病病原,可引起嚴(yán)重的皮膚感染,但目前常用的真菌性皮膚病的防治手段相對(duì)簡(jiǎn)單,對(duì)于該菌的研究仍大都停留在流行病學(xué)和藥物療效等方面,已不能滿足臨床醫(yī)學(xué)的需求。在真菌等微生物的生長(zhǎng)和繁殖中,鋅是一種重要的微量營(yíng)養(yǎng)元素,其常與多種功能蛋白結(jié)合以形成鋅指蛋白來(lái)完成生物學(xué)功能。目前,多種真菌的鋅吸收轉(zhuǎn)運(yùn)調(diào)控因子已經(jīng)明確,如:煙曲霉的ZafA蛋白、酵母菌的Zap1蛋白等,此類蛋白作為鋅響應(yīng)活力因子可在轉(zhuǎn)錄水平上對(duì)多種鋅轉(zhuǎn)運(yùn)蛋白的表達(dá)進(jìn)行調(diào)控,從而控制真菌對(duì)鋅離子的攝取,在真菌的生長(zhǎng)和致病過程中扮演重要角色。但目前,須癬毛癬菌的鋅響應(yīng)活力因子的基因組成及功能還未確定,鋅吸收轉(zhuǎn)運(yùn)調(diào)控網(wǎng)絡(luò)尚不清楚,確定須癬毛癬菌中鋅響應(yīng)活力因子以及鋅轉(zhuǎn)運(yùn)蛋白的基因組成對(duì)于須癬毛癬菌致病性的研究具有重要意義。研究發(fā)現(xiàn)多種真菌的鋅響應(yīng)活力因子的基因組成具有一定的相似性,初期試驗(yàn),以煙曲霉鋅響應(yīng)活力因子的基因序列為基礎(chǔ)設(shè)計(jì)引物,以須癬毛癬菌全基因組DNA、cDNA為模板進(jìn)行PCR擴(kuò)增,經(jīng)過多次試驗(yàn)發(fā)現(xiàn)無(wú)法獲得須癬毛癬菌鋅響應(yīng)活力因子的基因組成。因此,我們使用Illumina HiSeqTM 2000技術(shù)對(duì)正常狀態(tài)下及鋅缺乏情況下的須癬毛癬菌進(jìn)行了轉(zhuǎn)錄組測(cè)序,并對(duì)測(cè)序數(shù)據(jù)進(jìn)行了從頭組裝、基因功能注釋以及基因的差異表達(dá)分析等,后續(xù)通過BLASTX序列相似性搜索以及對(duì)須癬毛癬菌轉(zhuǎn)錄組測(cè)序數(shù)據(jù)的分析確定出須癬毛癬菌鋅響應(yīng)活力因子的基因組成,初步推斷出與鋅吸收轉(zhuǎn)運(yùn)相關(guān)的其他基因;并使用重組克隆技術(shù)構(gòu)建了針對(duì)ZafA基因的遺傳轉(zhuǎn)化用雙元載體,為后續(xù)的基因功能驗(yàn)證奠定了基礎(chǔ)。研究結(jié)果如下:1使用Illumina測(cè)序平臺(tái)對(duì)須癬毛癬菌進(jìn)行了RNA-seq,共獲得10751個(gè)unigenes,89%以上的基因得到功能注釋,對(duì)于須癬毛癬菌的基因功能研究以及基因組圖譜的組裝提供了寶貴的數(shù)據(jù)支持。2通過與煙曲霉ZafA基因、釀酒酵母Zap1基因進(jìn)行序列同源性比對(duì)以及功能注釋分析等,確定了須癬毛癬菌中與鋅吸收轉(zhuǎn)運(yùn)相關(guān)的基因并高保真擴(kuò)增出須癬毛癬菌ZafA基因。3將ZafA基因左右側(cè)翼片段以及來(lái)自質(zhì)粒pAN7-1的潮霉素B抗性基因(hph)同時(shí)插入到雙元載體pDHt/SK中,構(gòu)建ZafA基因轉(zhuǎn)化用雙元載體pDHt/ZafAⅠ、Ⅱ::hph,經(jīng)PCR及酶切驗(yàn)證,雙元載體構(gòu)建成功。試驗(yàn)結(jié)果表明,本研究完成了須癬毛癬菌的轉(zhuǎn)錄組測(cè)序及功能注釋;確定了須癬毛癬菌ZafA基因的組成,并成功地構(gòu)建了轉(zhuǎn)化用雙元載體pDHt/ZafAⅠ、Ⅱ::hph,為之后的ZafA基因的毒力試驗(yàn)及功能驗(yàn)證奠定了基礎(chǔ)。
[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，

本文編號(hào)：1639065