细菌水通道蛋白及其生理功能探讨

发布时间：2018-06-29 22:12

本文选题：简并PCR + RT-PCR　；参考：《重庆医科大学》2007年硕士论文

【摘要】： 第一部分:简并PCR探索10种细菌中的水通道蛋白目的: 用简并PCR方法扩增幽门螺旋杆菌、绿脓杆菌、乙型副伤寒杆菌、痢疾志贺菌、金黄色葡萄球菌、柠檬色葡萄球菌、白色葡萄球菌、脓肿分枝杆菌、大肠杆菌的水通道蛋白DNA,以探讨这10种细菌是否存在水通道蛋白,为进一步探讨水通道蛋白的生理功能奠定基础。方法: 以10种细菌全序列基因组DNA为模板,用细菌水通道蛋白的保守氨基酸序列设计细菌水通道蛋白的简并PCR通用引物,进行简并PCR扩增,将得到的目的片段进行PCR产物纯化、测序;BLAST比较基因序列,判定是否存在细菌水通道蛋白。结果: 简并PCR产物纯化、测序后在大肠杆菌中发现410bp大小序列,BLAST证实为水通道蛋白Glp-F。新发现在痢疾志贺菌中存在370bp大小序列,BLAST证实为水通道蛋白Glp-F。在其余8种细菌中未发现类似水通道蛋白的DNA。结论: 本试验证实简并PCR能够利用已知的水通道蛋白保守氨基酸序列扩增未知的DNA序列。探索到大肠杆菌存在水通道蛋白Glp-F,与国外报道一致,表明简并PCR能够探索细菌水通道蛋白的存在;新发现痢疾志贺菌中存在细菌水通道蛋白Glp-F,而国外报道在福氏志贺菌中存在细菌水通道蛋白Glp-F,推测志贺菌各群中可能都存在水通道蛋白;本试验的发现为探索水通道蛋白Glp-F生理作用奠定了基础。有8种细菌经简并PCR扩增未得到目的条带,表明水通道蛋白在细菌种群之间的分布存在不均一性。第二部分:高渗透压诱导水通道蛋白Glp-F在痢疾志贺菌内表达目的: 用渗透压不同的液体培养基和含有Hg2+(Glp-F选择性抑制剂)的不同渗透压培养基对痢疾志贺菌进行培养,诱导菌体内Glp-F的表达,以探讨Glp-F蛋白在菌体内的生理功能。方法: 1.配置不同渗透压培养基:普通牛肉浸液培养基;用无菌水稀释为含1/3、1/2牛肉浸液的培养基;加入NaCl使渗透浓度达到125mM、250mM、500mM、750mM培养基(N-原、N-1/3、N-1/2、N-125mM、N-250mM、N-500mM、N-750mM培养基)。 2.配置含有Hg2+抑制剂的不同渗透压培养基:在上述培养基中分别加入Glp-F的功能抑制剂HgCl22mg得到H-原、H-1/3、H-1/2、H-125mM、H-250mM、H-500mM、H-750mM培养基。 3.在上述各种培养基中接种痢疾志贺菌,分别于24,48,72,96小时培养点上观察细菌的生长情况,用紫外分光光度计测定其OD600值以评价细菌的生长情况。 4.用RT-PCR扩增Glp-F的mRNA,琼脂糖凝胶电泳、染色、成像分析,比较在不同环境条件下生长的痢疾志贺菌Glp-F的mRNA转录量。结果: 1.低渗透压环境中培养的痢疾志贺菌在各个观测点的生长无明显差异。 2.在N-培养基和H-培养基中生长情况出现显著不同:在H-培养基中细菌生长速度明显低于在N-培养基中生长速度,特别是在125mM、250mM浓度培养基中变化最大。 3. RT-PCR显示痢疾志贺菌Glp-F的mRNA含量在普通培养基、稀释1/3、1/2水的牛肉浸液培养基、N-500mM/750Mm、H- N-500mM/750Mm培养基中没有明显差异;在N-125mM /250mM培养基和H-125mM /250mM中含量明显增多,在H-125mM /250mM培养基中,细菌mRNA转录量大于在N-125mM/250mM培养基中的转录量。结论: Glp-F是细菌特有的水通道蛋白,探讨Glp-F的生理功能对研究细菌的水通蛋白有重要意义,国内尚未见报道。本试验的结果可以得到以下结论: 1.表达Glp-F蛋白的痢疾志贺菌在低渗透压下生长繁殖速度没有明显变化。 2.在高渗透压环境中,痢疾志贺菌的生长繁殖速度随着Glp-F蛋白功能的丧失而降低。 3. Glp-F的mRNA的在生长期内有不同的转录,对数生长期内尤为明显,与国外报道相似。 4.渗透压的升高能够诱导Glp-F的表达,尤见于N-125mM/250mM和H-125mM /250mM培养基中,并且各种渗透压培养基中水通道蛋白mRNA转录量依次为H-250mMH-125mMN-250mMN-125mM原始培养基。
[Abstract]:Part I: degenerate PCR explores aquaporins in 10 species of bacteria.
Objective:
A degenerate PCR method for amplification of Helicobacter pylori, Pseudomonas aeruginosa, paratyphus B, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus lemonade, Staphylococcus alba, Mycobacterium abscess, and water channel protein DNA of Escherichia coli to explore the presence of aquaporins for the further exploration of water channel proteins by these 10 bacteria. The physiological function lays the foundation.
Method:
10 bacteria full sequence genomic DNA was used as a template to design the degenerate PCR universal primers of bacterial aquaporin protein by the conservative amino acid sequence of bacterial aquaporin protein, and to degenerate and PCR amplification. The target fragments were purified and sequenced by PCR products, and BLAST was compared to determine whether there was a bacterial aquaporin protein.
Result:
The degenerate PCR product was purified and sequenced, and the 410bp size sequence was found in Escherichia coli. BLAST was proved to be a water channel protein Glp-F. found in Shigella dysenterias with 370bp size sequence, and BLAST confirmed that the aquaporin Glp-F. has not found a DNA. like aquaporin in the other 8 species of bacteria.
Conclusion:
This experiment confirmed that degenerate PCR can use the known amino acid sequence of the known aquaporin protein to amplify the unknown DNA sequence. To explore the existence of aquaporin Glp-F in Escherichia coli, it is consistent with foreign reports, indicating that degenerate PCR can explore the existence of bacterial aquaporin and the existence of bacterial aquaporin Glp-F in Shigella dysenterias. The existence of aquaporin Glp-F in Shigella flexneri was reported in foreign countries. It is suggested that there may be aquaporins in all Shigella groups. The discovery of this test laid the foundation for the exploration of the physiological role of water channel protein Glp-F. 8 kinds of bacteria have not been amplified by PCR and have not obtained the target bands, indicating that the aquaporin is in the bacterial population. The cloth exists inhomogeneity.
The second part: the expression of aquaporin Glp-F induced by high osmotic pressure in Shigella dysentery.
Objective:
Different osmotic media with different osmotic pressure and different osmotic pressure medium containing Hg2+ (Glp-F selective inhibitor) were used to culture Shigella dysentery and induce the expression of Glp-F in the bacteria in order to explore the physiological function of Glp-F protein in the bacteria.
Method:
1. different osmotic pressure medium: medium beef extract medium; diluted water as medium containing 1/3,1/2 beef extract; adding NaCl to reach 125mM, 250mM, 500mM, 750mM medium (N-, N-1/3, N-1/2, N-125mM, N-250mM, N-500mM, culture medium).
2. different osmotic pressure medium containing Hg2+ inhibitors: the function inhibitor HgCl22mg of Glp-F was added to the medium, respectively, to get H-, H-1/3, H-1/2, H-125mM, H-250mM, H-500mM, H-750mM medium.
3. inoculated Shigella dysenterias in the above medium, the growth of bacteria was observed at 24,48,72,96 hour culture point, and the value of OD600 was measured by ultraviolet spectrophotometer to evaluate the growth of bacteria.
4. the mRNA transcript of Glp-F of Shigella dysentery Glp-F was amplified by RT-PCR, agarose gel electrophoresis, staining and imaging analysis.
Result:
1. there was no significant difference in the growth of Shigella dysentery cultured at different observation points in low osmotic pressure.
2. there were significant differences in the growth of N- medium and H- medium: the growth rate of bacteria in the H- medium was significantly lower than that in the N- medium, especially in the 125mM and 250mM medium.
3. RT-PCR showed that the mRNA content of Shigella dysentery Glp-F was in the ordinary medium, the medium of diluted 1/3,1/2 water, the medium of N-500mM/750Mm, H- N-500mM/750Mm, and the content of the N-125mM /250mM medium and H-125mM /250mM increased. The amount of transcription in the /250mM medium.
Conclusion:
Glp-F is a specific water channel protein of bacteria. It is of great significance to explore the physiological function of Glp-F for the study of bacteria's water protein. The results of this experiment can be concluded as follows:
1. the growth rate of Shigella dysentery expressing Glp-F protein did not change significantly under low osmotic pressure.
2. in the hyperosmotic environment, the growth and reproduction rate of Shigella dysentery decreased with the loss of Glp-F protein function.
3. mRNA of Glp-F has different transcripts during the growth period, especially in the logarithmic growth phase, which is similar to that reported abroad.
The increase of 4. osmotic pressure can induce the expression of Glp-F, especially in N-125mM/250mM and H-125mM /250mM medium, and the transcription of water channel protein mRNA in various osmotic media is in turn H-250mMH-125mMN-250mMN-125mM original medium.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378.25

【共引文献】