新西兰匍柄霉（Stemphylium eturmiunum）中与ASF1，STE2和STE3互作蛋白的筛选

发布时间：2018-06-06 01:48

本文选题：新西兰匍柄霉 + 酵母双杂交　；参考：《山东农业大学》2017年硕士论文

【摘要】：匍柄霉属(Stemphylium)是丝状子囊真菌中的一类重要真菌类群,在自然界中该属真菌绝大多数以无性态形式存在,但也有一小部分存在有性生殖阶段,而新西兰匍柄霉(Stemphylium eturmiunum)就是其中能产生有性态的种类之一。为了研究新西兰匍柄霉(S.eturmiunum)与其它匍柄霉属种在有性态进化方面的区别,我们查阅资料发现真菌的有性进化受若干性别相关因子的调控。研究表明,信息素受体基因STE2和STE3在模式菌酿酒酵母、粗糙脉孢菌等菌中都有调控有性发育通路的作用。此外,反沉默因子ASF1对有性发育也有着一定的调控作用。研究表明,△STE2、△STE3和△ASF1单突变菌株在适宜的环境下培养均不能产生有性态。真菌异宗配合发生在两种细胞类型:a和α之间,细胞类型由分泌的信息素控制。两种类型细胞分别能产生两种信息素前体a和α,及两种相应的信息素受体STE2和STE3。在有性生殖过程中,经过加工的信息素α因子被a细胞表面的STE2受体识别,启动交配信号;同样,经过加工的信息素a因子被α细胞表面的STE3受体识别,启动交配信号。为了进一步探索敲除信息素受体基因STE2和STE3后,是否影响其它上下游基因的表达,我们以新西兰匍柄霉(S.eturmiunum)为实验材料,主要进行了如下研究:利用Trizol法分别对△STE2和△STE3单突变菌株提取总RNA并进行转录组测序,结果表明敲除信息素受体基因STE2或STE3会影响其上下游一系列基因的正常转录。经过分析与预测,我们从中选取了一系列差异表达比较大的基因,试图筛选信息素受体STE2和STE3的互作蛋白。通过基因序列的比对,我们发现STE2和STE3基因各含有一个内含子。我们通过Over Lap PCR获得了没有内含子的基因序列,并在随后的酵母双杂实验过程中,发现信息素受体STE2和ORF07010(糖苷水解酶家族32蛋白)能够相互作用。反沉默因子ASF1(anti-silencing function 1)在进化上非常保守,而且是组蛋白H3/H4的分子伴侣,在基因转录、DNA复制和修复等多个染色质层面的生物学过程中发挥着重要的作用。通过同源序列比对,我们发现新西兰匍柄霉(S.eturmiunum)基因组中含有ASF1基因。根据已报到的ASF1基因的信息,设计特异性引物克隆了新西兰匍柄霉(S.eturmiunum)中的反沉默因子ASF1和组蛋白H3、H4的基因。同时利用酵母双杂以及Pull-down实验技术证明,ASF1确实能与组蛋白H4互作,但与组蛋白H3不存在相互作用,这可能与其特定的功能相关。新西兰匍柄霉(S.eturmiunum)中信息素受体△STE2和△STE3单缺突变菌株的转录组测序以及反沉默因子Asf1互作蛋白的初步研究,为深入研究新西兰匍柄霉中这三种蛋白的结构与功能打下了良好的基础,为深入探索生物性别起源与分化历程提供了信息积累。
[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 伪 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 伪 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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q933

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