论文总字数：23556字

摘 要

曲霉中与高等动、植物类似，存在保守且独特的钙信号系统。钙通道分布于质膜、内质网膜、液泡膜和高尔基体膜上，通过响应外界压力刺激，介导外钙内流、胞质钙流向钙库或钙库钙释放等，从而维持细胞的钙稳态，使细胞进行正常生长与代谢。近年来，钙离子对于生物膜的影响是研究的热门领域，钙通道基因通过影响细胞壁的组分合成来调控生物膜的形成，又由于生物膜的形成和固定化发酵效能直接相关，所以研究钙通道基因和生物膜形成的机制对于发酵工业生产有重要意义。

本课题以黑曲霉为模式生物，通过数据库分析确证了两种酿酒酵母ScYvc1和ScSpf1同源的蛋白YvcA和SpfA，钙通道YvcA定位于液泡膜上，而SpfA则是维持内质网高钙水平的钙泵。对这两种钙通道以同源重组方法进行基因敲除，以期研究其对黑曲霉生物膜形成和固定化发酵的影响。通过SDS、CFW生物膜抗逆性实验和结晶紫染色实验，发现△YvcA和△SpfA两种敲除菌的生物膜形成能力均减弱，推测其与黑曲霉细胞壁中的几丁质和葡聚糖等多糖缺失有关。通过固定化发酵实验发现敲除菌的耗糖量和产酸量比原始菌低，在游离发酵时敲除菌和原始菌耗糖量和产酸量相差不大，从侧面说明了生物膜对固定化发酵的重要性。

关键词：黑曲霉 钙通道 基因敲除 固定化 生物膜

Gene knockout of calcium channel YvcA and SpfA in Aspergillus nigra and its effect on curing biofilm formation

ABSTRACT

Aspergillus has a conservative and unique calcium signal system, which is similar to high animal and plant. Calcium channels are distributed on plasma membrane, endoplasmic reticulum, vacuole membrane and Golgi membrane. Calcium homeostasis is maintained by mediating external calcium influx and cytoplasmic calcium flow to calcium pool or calcium release in response to external pressure stimulation. Make the cell grow and metabolize normally. In recent years, the effect of calcium ion on biofilm is a hot research field. Calcium channel gene regulates the formation of biofilm by affecting the synthesis of cell wall components, and the formation quality of biofilm is directly related to the efficiency of immobilization and fermentation. Therefore, it is of great significance to study the mechanism of calcium channel gene and biofilm formation in fermentation industry.

In this study, Aspergillus nigra was used as a model organism. Through database analysis, it was confirmed that two kinds of Saccharomyces cerevisiae ScYvc1 and ScSpf1 homologous proteins YvcA and SpfA, calcium channel YvcA were located on vacuole membrane, while SpfA was a calcium pump to maintain high calcium level in ER. Through the stress resistance test and crystal purple staining test of SDS,CFW biofilm, it was found that the biofilm formation ability of YvcA and SpfA knockout bacteria was weakened, which was speculated to be related to the lack of polysaccharides such as chitin and dextran in the cell wall of Aspergillus nigra. The results of immobilization fermentation showed that the sugar consumption and acid production of knockout bacteria were lower than those of original bacteria, and there was no significant difference between the sugar consumption and acid production of knockout bacteria and original bacteria in free fermentation, which explained the importance of biofilm to immobilization fermentation.

Key words: Aspergillus nigra; calcium channel; gene knockout; cell immobilization; biofilm

目录

摘要 I

ABSTRACT II

目录 I

第一章 前言 1

1.1 选题背景 1

1.2 模式生物黑曲霉 2

1.3 钙信号系统 2

1.3.1 钙稳态成员 2

1.3.2 钙信号途径 3

1.3.3 钙通道基因YvcA和SpfA的功能研究 4

1.4 真菌生物膜 5

1.4.1生物膜的形成过程 5

1.4.2 曲霉生物膜的研究进展 6

1.5 固定化发酵 7

1.5.1细胞固定化技术 7

1.5.2 固定化发酵的应用 8

1.6 设计思路 8

第二章 实验部分 10

2.1菌株和质粒 10

2.2实验仪器及药品 10

2.3缓冲液 12

2.4 黑曲霉的活化 12

2.4.1 培养基 12

2.4.2 实验方法 12

2.5 YvcA和SpfA的基因敲除 13

2.5.1设计引物 13

2.5.2 片段扩增 14

2.5.3 三段融合 15

2.5.4 黑曲霉原生质体制备与转化 15

2.6 黑曲霉生物膜抗逆性实验 16

2.6.1 培养基 16

2.6.2 实验方法 16

2.7 黑曲霉结晶紫染色实验 16

2.7.1 培养基 16

2.7.2 实验方法 17

2.8 固定化发酵 17

2.8.1 培养基 17

2.8.2 发酵方法 17

2.8.3 高效液相色谱参数 18

第三章 结果与分析 19

3.1 黑曲霉YvcA和SpfA的基因敲除 19

3.2 生物膜抗逆性实验 21

3.3 结晶紫染色实验验证 22

3.4 固定化发酵 24

第四章 结论与展望 28

4.1 结论 28

4.2 展望 28

参考文献 30

致谢 33

第一章 前言

1.1 选题背景

黑曲霉是目前工业上广泛使用的发酵菌种，能利用木糖、葡萄糖等糖类经过EMP和HMP途径产生丙酮酸，丙酮酸后续转化为乙酰CoA和草酰乙酸，两者缩合得到柠檬酸。

柠檬酸是黑曲霉有机酸生产的代表之一，在食品工业中被誉为“第一食用酸味剂”，广泛地用作饮料、糕点、豆制品等的缓冲剂、pH调节剂和抗氧化剂等。另外，在化工和医药工业领域，柠檬酸是重要的多价螯合剂，同时也是化学中间体，兼具媒染和洗涤金属等功效。

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