论文总字数：18441字

摘 要

微生物生物膜可以被定义为附着在表面以及嵌入细胞外基质（ECM）的多细胞聚集体。非致病性酵母，如酿酒酵母，都有如下微生物生物膜的共同特点，即细胞—细胞和细胞—表面的粘附。酿酒酵母能产生ECM，且能够响应群体感应。其多细胞聚集体对抗真菌药物不敏感。附着效应是由一类细胞表面蛋白调控的，其中Fbp1已被证明是合成生物膜必不可少的物质。Fbp1表达通过一系列调控途径的调控，包括蛋白激酶A和有丝分裂原活化蛋白激酶途径。目前已经开发了一系列研究酿酒酵母的先进的基因工具和资源，包括在一系列产膜菌株为背景的基因敲除菌和GFP融合蛋白。此外，激光共聚焦扫描显微镜和蛋白质，DNA和RNA荧光标记也经常用于生物膜研究。这些技术可以用于揭示对于生物膜形成的分子机理，耐药性以及对分子间的相互作用，细胞响应环境影响，不同细胞间的区别和壁龛在酿酒酵母生物膜。由于其与念珠菌属密切相关，酿酒酵母被作为研究致病性酵母的一种模型。

为了研究和生物膜有关的基因的生物学功能，接下来我们通过分子生物学手段对调控生物膜形成的关键基因进行分子改造和基因修饰。利用长同源臂同源重组（LFH-PCR）技术，我们成功的构建了工业双倍体酿酒酵母1306的Fbp1单基因敲除菌1306-FD、Fks1单基因敲除菌1306-KD以及Fks1双基因敲除菌1306-M，并对酵母野生菌和敲除菌进行细胞生长、成膜形态、invasive分析、固定化细胞乙醇敏感性等方面的分析。

关键词：酿酒酵母生物膜 转录组分析 同源重组敲除

Abstract

Microbial biofilms can be defined as multi-cellular aggregates adhering to a surface and embedded in an extracellular matrix (ECM). The nonpathogenic yeast, Saccharomyces cerevisiae, follows the common traits of microbial biofilms with cell–cell and cell–surface adhesion. S. cerevisiae is shown to produce an ECM and respond to quorum sensing, and multi-cellular aggregates have lowered susceptibility to antifungals. Adhesion is mediated by a family of cell surface proteins of which Fbp1 has been shown to be essential for biofilm development. Fbp1 expression is regulated via a number of regulatory pathways including the protein kinase A and a mitogen-activated protein kinase pathway. Advanced genetic tools and resources have been developed for S. Cerevisiae including a deletion mutant-strain collection in a biofilm-forming strain background and GFP-fusion protein collections. Furthermore, S. Cerevisiae biofilm is well applied for confocal laser scanning microscopy and fluorophore tagging of proteins, DNA and RNA. These techniques can be used to uncover the molecular mechanisms for biofilm development, drug resistance and for the study of molecular interactions, cell response to environmental cues, cellto cell variation and niches in S. cerevisiae biofilm. Being closely related to Candida species, S. cerevisiae is a model to investigate biofilms of pathogenic yeast.

To study the biological functions of genes related to biofilms, we employed molecular biology methods to research the biofilm genes’ functions. Using the Homologous recombination technology to construct Fbp1 and Fks1 knockout strain 1306-FD, 1306-KD, 1306-M. later, the difference of wild stain 1306 and Fbp1/Fks1 gene knockout stains were studied with the some methods such as growth curve, biofilm morphology, invasive analysis and ethanol sensitivity analysis. Results showed that yeast 1306-FD formed the defective biofilm, which is consistent with the previous reports, and the ability of forming biofilm was decline. At the same time, we also found that like the yeast 1306-FD, 1306-KD and 1306-M knockout strains can’t form completed biofilms .

KEYWORD: Saccharomyces cerevisiae biofilms; Transcriptional analysis; Homologous recombination knockout

目 录

摘 要 I

Abstract II

第一章 文献综述 5

1.1 概述 5

1.2 酵母菌生物膜 5

1.2.1 生物膜形成过程 5

1.2.2 生物膜结构及功能 6

1.2.3调控生物膜形成相关基因 7

1.3 生物膜在工业化生产应用 7

1.4 论文立题背景及主要研究内容 8

1.4.1 选题背景 8

1.4.2 研究内容 8

第二章 生物膜形成 调控基因的敲除 9

2.1 材料与试剂 9

2.1.1 菌种及引物 9

2.1.2 实验试剂 9

2.1.3 实验仪器 10

2.2 培养及组成 10

2.3 实验方法 11

2.3.1 酵母菌培养 11

2.3.2 基因敲片构建 11

2.3.3 电转 14

2.4 结果与讨论 14

2.5 小结 15

第三章 基因敲除菌表型及乙醇耐受性 15

3.1 材料与试剂 15

3.1.1 菌种及引物 15

3.1.2 实验试剂 15

3.1.3 仪器 15

3.2 试验方法 15

3.2.1 絮凝实验 15

3.2.2 附着实验 15

3.2.3 乙醇耐受性实验 15

3.3 结果与讨论 16

3.4 小结 21

第四章 结论和展望 22

4.1 结论 22

4.2 展望 22

参考文献 23

致谢 23

第一章 文献综述

1.1 概述

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