| 产碱杆菌CzcD基因的克隆，表达以及功能的初步研究 |
| Alternative Title | Cloning, expression and functional analysis of Alcaligenes eutrophus CH34 CzcD gene
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| 毛娟 |
| Thesis Advisor | 谢小冬
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| 2008-05-24
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| Degree Grantor | 兰州大学
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| Place of Conferral | 兰州
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| Degree Name | 硕士
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| Keyword | Alcaligenes eutrophus CH34
CzcD基因
重金属抗性
质粒消除
转化
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| Abstract | CzcD基因位于产碱杆菌(Alcaligenes eutrophus) CH34的质粒pMLO30上，研究表明：该基因编码的蛋白质是Alcaligenes eutrophus CH34中czc离子泵出系统中的一员，该系统主要通过CzcA，B，C成员完成对重金属的泵出、转运等过程，从而增强菌株对某些重金属的抗性。而CzcD对CzcA，B，C的表达有诱导作用。本论文以产碱杆菌菌株CH34的质粒为模板，经PCR扩增得到CzcD基因；并转化大肠杆菌BL21菌株，研究其诱导表达情况。同时，为了检测蛋白质CzcD是否具有一定的重金属抗性，通过设计相应的阴阳性对照，将各种菌株在不同浓度的重金属培养基中培养，检测OD600值以观察各细菌的生长状况，发现在CzcD蛋白质的作用下，转入CzcD基因的工程菌较野生型菌株的生长量大，具有相对较强的重金属抗性。本文为研究细菌对重金属的抗性机理，以及构建具有重金属抗性的工程菌来高效净化环境中重金属的污染提供一定的理论基础。
本论文研究得到以下结果：
1.PCR扩增得到了CzcD基因，将其转化大肠杆菌BL21菌株，得到PETCZCD菌株。当该菌液的OD600值达到1.0，培养温度为37℃时，在终浓度为1.0 mM的IPTG诱导下，蛋白有较高的表达量。
2.得到不同类型的阴，阳性对照菌株：对产碱杆菌CH34的野生菌做质粒消除实验得到菌株△CH34。PETCZCD菌株提取质粒转化到△CH34中得到菌株JH。pET-28(a)空载体转化大肠杆菌BL21得到菌株PETBL21。
3.CzcD蛋白质能赋予宿主菌一定的重金属钴，锌，镉的抗性。 |
| Other Abstract | The CzcD gene was located in the plasmid pMOL30 of Alcaligenes eutrophus CH34. The research have revealed that the protein CzcD is one of the czc iron efflux system in the Alcaligenes eutrophus CH34 and it is an important inducer to the expression of CzcA, B, C. The czc system can pump and transport some heavy metal iron by protein CzcA, B, C. In this paper, CzcD gene was cloned and the product was transformed into Escherichia coli BL21 to study the expression of CzcD in the host. Whether or not the CzcD had the heavy metal resistance was also been studied in this paper. Different positive and negative controls were gained. All the strains grew in the medium with different heavy metal to see the growth difference between them. This research supplies some rationale to the study of the bacterium heavy metal resistance and the construction of the new engineering strains which have the ability to clean the heavy metal pollution in the environment.
The results were as follows:
1. CzcD gene was cloned and transformed into Escherichia coli BL21 to gain the strain PETCZCD. And abundance of protein CzcD was gotten after PETCZCD was cultivated in the LB for 4 hours at 37 ℃ when the OD600 was at 1.0, adding 1.0mM IPTG.
2. Different positive and negative controls were gained. The △CH34 which was
obtained by plasmid curing of wildtype Alcaligenes eutrophus CH34. Then the plamid of PETCZCD was transformed into △CH34 and the strain was named JH.
3. The protein CzcD could generate resistance to heavy metal itself. |
| URL | 查看原文
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| Language | 中文
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| Document Type | 学位论文
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| Identifier | https://ir.lzu.edu.cn/handle/262010/221638
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| Collection | 生命科学学院
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Recommended Citation
GB/T 7714 |
毛娟. 产碱杆菌CzcD基因的克隆，表达以及功能的初步研究[D]. 兰州. 兰州大学,2008.
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