本文選題：煙曲霉 + Bem46��； 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:細(xì)胞的極性生長是指從細(xì)胞的某一區(qū)域非對稱性的生長出另一種特定的結(jié)構(gòu)或形狀。這種獨(dú)特的結(jié)構(gòu)對細(xì)胞的功能至關(guān)重要,并協(xié)助細(xì)胞間的相互作用。煙曲霉等曲霉菌屬是典型的極性生長生物體,主要表現(xiàn)為其菌絲尖端的不斷生長。本文將構(gòu)建煙曲霉Bem46基因敲除株,并在配制含不同抗真菌藥物的培養(yǎng)基上,觀察煙曲霉Bem46基因缺陷株與對照株的生長差異,以期發(fā)現(xiàn)新的藥物靶點(diǎn);以及構(gòu)建煙曲霉Bem46基因熒光定位株,觀察其在煙曲霉菌絲中的定位情況;對比對照株及敲除株發(fā)芽狀況并檢測部分Bem46極性生長傳導(dǎo)通路中的相關(guān)基因表達(dá)量情況來探索Bem46基因在煙曲霉極性生長過程中的作用。方法:生物信息學(xué)方法查找煙曲霉中Bem46基因,設(shè)計引物,并以pyrG作為篩選標(biāo)記構(gòu)建煙曲霉Bem46基因敲除株(ΔBem46)。在基礎(chǔ)培養(yǎng)基接種相同數(shù)量的對照株與缺陷株孢子,觀察二者生長有無差異。配制五種藥物(兩性霉素B、卡泊芬凈、伊曲康唑、伏立康唑、他克莫司)不同濃度的平皿,濃度分別為0、0.1、0.3、0.5、0.8、1.0、2.0、5.0mg/L,點(diǎn)種相同數(shù)量的對照株及敲除株的孢子,37℃培養(yǎng)72h,測量并記錄直徑,實驗重復(fù)三次。將Bem46基因與增強(qiáng)型綠色熒光蛋白(EGFP)及潮霉素篩選轉(zhuǎn)化子相連,轉(zhuǎn)入煙曲霉,潮霉素篩選后得到Bem46熒光定位株,在共聚焦顯微鏡下觀察Bem46在煙曲霉極性生長過程中的定位情況。在倒置顯微鏡下對比敲除株與對照株的出芽差異,然后分別提取二者的RNA,使用實時熒光定量PCR方法檢測極性生長相關(guān)基因Bem1、Bud5在對照株及敲除株中的表達(dá)差異。結(jié)果:通過序列比對在煙曲霉基因組中找到了Bem46基因,其編號為Afu7g04660,由1116bp堿基組成,編碼311個氨基酸。經(jīng)PCR和Southernblot驗證得到Bem46基因敲除株。在五種藥物不同濃度平皿上,對照株與敲除株生長直徑經(jīng)重復(fù)測量方差分析并無明顯差異。經(jīng)原生質(zhì)體轉(zhuǎn)化法及PCR驗證成功得到Bem46基因熒光定位株,在共聚焦顯微鏡下觀察,熒光在菌絲內(nèi)呈彌散分布,并沒有觀察到特異性的定位區(qū)域,但在分支處有聚集現(xiàn)象。敲除株與定位株的出芽率經(jīng)重復(fù)測量方差分析,可見敲除株出芽明顯滯后于對照株。極性生長相關(guān)基因Bem1、Bud5經(jīng)實時熒光定量PCR檢測,在敲除株中的表達(dá)量均低于對照株。結(jié)論:煙曲霉Bem46基因并不能作為兩性霉素B、卡泊芬凈、伊曲康唑、伏立康唑、他克莫司五種藥物的藥物靶點(diǎn);Bem46基因并不能直接構(gòu)成極性生長的相關(guān)結(jié)構(gòu),如菌絲隔膜或菌絲尖端等;但Bem46基因確實參與煙曲霉的極性生長過程,該基因可能有促進(jìn)孢子發(fā)芽的作用,并且它的缺失導(dǎo)致極性生長相關(guān)基因表達(dá)下調(diào),極有可能影響了極性生長傳導(dǎo)通路,為研究煙曲霉生長機(jī)制提供了新思路。
[Abstract]:Objective: polar growth of cells refers to the asymmetric growth of a cell from one region of another specific structure or shape. This unique structure is essential for cell function and facilitates intercellular interaction. Aspergillus such as Aspergillus fumigatus is a typical polar growth organism, which is mainly characterized by the continuous growth of its hyphae tip. In this paper, the Bem46 gene knockout strain of Aspergillus fumigatus was constructed, and the growth difference between Bem46 gene deficient strain of Aspergillus fumigatus and control strain was observed on the medium containing different antifungal drugs, in order to find new drug target. The fluorescent locus of Bem46 gene of Aspergillus fumigatus was constructed and its localization in Aspergillus fumigatus filaments was observed. To explore the role of Bem46 gene in polar growth of Aspergillus fumigatus, the germinating status of control plants and knockout plants was compared and the expression of related genes in some Bem46 polar growth conduction pathways was detected. Methods: Bem46 gene in Aspergillus fumigatus was identified by bioinformatics, primers were designed, and pyrG was used as screening marker to construct Bem46 knockout plant (螖 Bem46C) of Aspergillus fumigatus. The spores of the control and defective strains were inoculated in the basic culture medium to observe the difference between them. Five kinds of drugs (amphotericin B, carpofensin, itraconazole, volconazole, tacrolimus) were prepared with different concentrations of 0 0. 1 ~ 0. 3 ~ 0. 5 ~ 0. 0 mg 路L ~ (-1) and 0. 8% 0. 0 mg 路L ~ (-1). The spores of the control and knockout plants were cultured at 37 鈩，

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