本文選題：新西蘭匍柄霉 + 酵母雙雜交　； 參考：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：匍柄霉屬(Stemphylium)是絲狀子囊真菌中的一類重要真菌類群,在自然界中該屬真菌絕大多數(shù)以無性態(tài)形式存在,但也有一小部分存在有性生殖階段,而新西蘭匍柄霉(Stemphylium eturmiunum)就是其中能產(chǎn)生有性態(tài)的種類之一。為了研究新西蘭匍柄霉(S.eturmiunum)與其它匍柄霉屬種在有性態(tài)進化方面的區(qū)別,我們查閱資料發(fā)現(xiàn)真菌的有性進化受若干性別相關(guān)因子的調(diào)控。研究表明,信息素受體基因STE2和STE3在模式菌釀酒酵母、粗糙脈孢菌等菌中都有調(diào)控有性發(fā)育通路的作用。此外,反沉默因子ASF1對有性發(fā)育也有著一定的調(diào)控作用。研究表明,△STE2、△STE3和△ASF1單突變菌株在適宜的環(huán)境下培養(yǎng)均不能產(chǎn)生有性態(tài)。真菌異宗配合發(fā)生在兩種細(xì)胞類型:a和α之間,細(xì)胞類型由分泌的信息素控制。兩種類型細(xì)胞分別能產(chǎn)生兩種信息素前體a和α,及兩種相應(yīng)的信息素受體STE2和STE3。在有性生殖過程中,經(jīng)過加工的信息素α因子被a細(xì)胞表面的STE2受體識別,啟動交配信號;同樣,經(jīng)過加工的信息素a因子被α細(xì)胞表面的STE3受體識別,啟動交配信號。為了進一步探索敲除信息素受體基因STE2和STE3后,是否影響其它上下游基因的表達(dá),我們以新西蘭匍柄霉(S.eturmiunum)為實驗材料,主要進行了如下研究:利用Trizol法分別對△STE2和△STE3單突變菌株提取總RNA并進行轉(zhuǎn)錄組測序,結(jié)果表明敲除信息素受體基因STE2或STE3會影響其上下游一系列基因的正常轉(zhuǎn)錄。經(jīng)過分析與預(yù)測,我們從中選取了一系列差異表達(dá)比較大的基因,試圖篩選信息素受體STE2和STE3的互作蛋白。通過基因序列的比對,我們發(fā)現(xiàn)STE2和STE3基因各含有一個內(nèi)含子。我們通過Over Lap PCR獲得了沒有內(nèi)含子的基因序列,并在隨后的酵母雙雜實驗過程中,發(fā)現(xiàn)信息素受體STE2和ORF07010(糖苷水解酶家族32蛋白)能夠相互作用。反沉默因子ASF1(anti-silencing function 1)在進化上非常保守,而且是組蛋白H3/H4的分子伴侶,在基因轉(zhuǎn)錄、DNA復(fù)制和修復(fù)等多個染色質(zhì)層面的生物學(xué)過程中發(fā)揮著重要的作用。通過同源序列比對,我們發(fā)現(xiàn)新西蘭匍柄霉(S.eturmiunum)基因組中含有ASF1基因。根據(jù)已報到的ASF1基因的信息,設(shè)計特異性引物克隆了新西蘭匍柄霉(S.eturmiunum)中的反沉默因子ASF1和組蛋白H3、H4的基因。同時利用酵母雙雜以及Pull-down實驗技術(shù)證明,ASF1確實能與組蛋白H4互作,但與組蛋白H3不存在相互作用,這可能與其特定的功能相關(guān)。新西蘭匍柄霉(S.eturmiunum)中信息素受體△STE2和△STE3單缺突變菌株的轉(zhuǎn)錄組測序以及反沉默因子Asf1互作蛋白的初步研究,為深入研究新西蘭匍柄霉中這三種蛋白的結(jié)構(gòu)與功能打下了良好的基礎(chǔ),為深入探索生物性別起源與分化歷程提供了信息積累。
[Abstract]:Stemphylium is an important group of filamentous ascomycetes. In nature, most of the fungi of the genus Stemphylium exist in the form of asexual, but there is also a small number of them in the stage of sexual reproduction. Stemphylium eturmiunum of New Zealand is one of the species which can produce sexual state. In order to study the difference between S.eturmiunumum and other species in sexual evolution, we found that the sexual evolution of fungi is regulated by several sex related factors. The results showed that the pheromone receptor genes STE2 and STE3 could regulate the sexual development pathway in Saccharomyces cerevisiae and C. crassa. In addition, anti-silencing factor ASF1 also plays a regulatory role in sexual development. The results showed that STE _ 2, STE3 and ASF1 single mutant strains could not produce sexual state under suitable conditions. Fungal heterogeneity occurs between two cell types: a and a, and the cell types are controlled by secreted pheromones. Two types of cells produced two pheromone precursors a and a, and two corresponding pheromone receptors, STE2 and STE3, respectively. During sexual reproduction, the processed pheromone 偽 factor is recognized by the STE2 receptor on the surface of a cell and the mating signal is activated. Similarly, the processed pheromone a factor is recognized by the STE3 receptor on the 偽 cell surface and the mating signal is initiated. In order to further explore whether knockout of pheromone receptor genes STE2 and STE3 could affect the expression of other upstream and downstream genes, S.eturmiunum of New Zealand was used as the experimental material. The results showed that knockout of the pheromone receptor gene STE2 or STE3 affected the normal transcription of a series of upstream and downstream genes. After analysis and prediction, we selected a series of differentially expressed genes and tried to screen the interaction proteins of pheromone receptor STE2 and STE3. By the alignment of gene sequence, we found that STE2 gene and STE3 gene each contain an intron. We obtained the gene sequence without intron by Over Lap PCR and found that the pheromone receptor STE2 and ORF07010 (glycoside hydrolase family 32 protein) could interact with each other in the subsequent yeast double hybrid experiment. Anti-silencing factor (ASF1(anti-silencing function 1) is very conserved in evolution and is a molecular companion of histone H3/H4, which plays an important role in the biological processes of multiple chromatin levels such as gene transcription, DNA replication and repair. By homologous sequence alignment, we found that the genome of S. Eturmiunum contained ASF1 gene. Based on the information of reported ASF1 gene, a specific primer was designed to clone the genes of anti-silencing factor ASF1 and histone H3H4 in S.eturmiunum. At the same time, yeast double hybrids and Pull-down techniques were used to prove that ASF1 could interact with histone H4, but not with histone H3, which might be related to its specific function. Transcriptome sequencing of pheromone receptor STE2 and STE3 mono-deficient mutant strains and preliminary study on anti-silencing factor Asf1 interaction protein in S.eturmiunum, New Zealand. These three proteins laid a good foundation for further study on the structure and function of these three proteins, and provided information accumulation for further exploring the origin and differentiation of biological sex.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q933

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