ICT1基因敲除对酵母菌耐盐性的影响
ICT1基因敲除对酵母菌耐盐性的影响(任务书,开题报告,外文翻译,论文8000字)
摘 要
酵母在生产应用过程中经常遇到各种应激环境如高温高渗透压等，其中在高盐环境易下，容易导致失水过多而死亡，影响发酵后续，所以对酵母的耐盐性进行初步探讨具有一定的实际意义。本文以BY4741作为出发菌株，利用基因敲除构建出△ICT1重组株。通过盐浓度稀释点样研究其耐盐表型，对不同盐浓度下麦角固醇含量进行测定发现△ICT1重组菌经NaCl处理后只能合成少量的麦角固醇，相比于正常条件下麦角固醇含量下降了66.9%。同时，还利用PI染色法测定了细胞膜完整性，并用荧光显微镜观察染色情况，△ICT1重组菌大多数细胞被染成红色，细胞膜受损严重。最后对膜上转运蛋白基因NHA1和ENA1转录水平进行了Q-PCR测定，进一步验证了ICT1的敲除可能也会通过下调离子转运蛋白基因的转录水平，降低质膜的离子转运能力，最终导致酵母耐盐性的下降。
关键词：酵母菌 基因敲除 耐盐性 ICT1
Effect of ICT1 gene knockout on salt tolerance of yeast
ABSTRACT
Yeast often encounters various stress environments such as high temperature and high osmotic pressure in the production and application process. Among them, in the high salt environment, it easily leads to excessive water loss and death, affecting the subsequent fermentation, so the salt tolerance of yeast The preliminary discussion has a certain practical significance. In this study, BY4741 was used as the starting strain, and the recombinant strain ICT1 was constructed by knockout. The salt-tolerance phenotype was studied by diluting samples with salt concentration. The ergosterol content was determined at different salt concentrations. It was found that the ΔICT1 recombinant strain could only synthesize a small amount of ergosterol after NaCl treatment, compared to normal conditions. Ergosterol content decreased by 66.9%. At the same time, the integrity of the cell membrane was measured by PI staining, and the staining was observed by fluorescence microscopy. Most cells of the ICT1 recombinant strain were stained red and the cell membrane was severely damaged. Finally, a Q-PCR assay was performed to determine the transcription levels of the transporter genes NHA1 and ENA1 on the membrane. This confirms that the knockdown of ICT1 may also result in down-regulation of the ion transporter gene transcription level and decrease the ion transport capacity of the plasma membrane. Decline in yeast salt tolerance.
Key words: yeast gene knockout salt tolerance ICT1
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目录
摘 要 I
ABSTRACT II
第一章 文献综述 1
1.1 酿酒酵母的耐性机制 1
1.1.1 金属离子的动态平衡对酵母菌耐盐性的影响[1] 1
1.1.2 相关基因的表达 1
1.1.3 细胞表面的脂质变化 1
1.2 基因敲除技术的研究进展 2
1.3 酵母菌耐盐性研究进展 3
第二章 实验部分 4
2.1 实验材料 4
2.1.1 菌种与质粒 4 [资料来源：http://Doc163.com]
2.1.2 主要工具酶与试剂盒表 4
2.1.3 主要试剂 5
2.1.4 主要仪器 5
2.1.5 培养基 6
2.2 实验方法 6
2.2.1 pUG6质粒的提取 6
2.2.2 ICT1基因敲除盒的引物设计 6
2.2.3 ICT1基因敲除盒的构建 7
2.2.4 阳性转化子验证 7
2.2.5 酵母电转化感受态的制备 8
2.2.6 酵母的电转化 9
2.2.7 不同NaCl浓度的稀释点滴实验 10
2.2.8 PI染色测定膜完整性 10
2.2.9 荧光定量PCR的测定 10
第三章 结果与讨论 11
3.3 结果与讨论 11
3.3.1 ICT1基因敲除盒的验证 11
3.3.2 ICT1基因敲除组件的电转化 11
3.3.3 转化子的筛选验证 12
3.3.4 不同盐浓度下BY4741和重组菌△ICT1的稀释点滴实验 13
3.3.5 BY4741和△ICT1重组菌在不同盐浓度下麦角固醇含量的测定 14
3.3.6 荧光显微镜测定膜完整性 15
3.3.7 荧光定量PCR测定酵母细胞膜上转运蛋白基因的转录水平 15
第四章 结论与展望 17
4.1 结论 17
4.2 展望 17
参考文献 18
致谢 19