本文選題：簡(jiǎn)并PCR + RT-PCR　； 參考：《重慶醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 第一部分:簡(jiǎn)并PCR探索10種細(xì)菌中的水通道蛋白 目的: 用簡(jiǎn)并PCR方法擴(kuò)增幽門螺旋桿菌、綠膿桿菌、乙型副傷寒桿菌、痢疾志賀菌、金黃色葡萄球菌、檸檬色葡萄球菌、白色葡萄球菌、膿腫分枝桿菌、大腸桿菌的水通道蛋白DNA,以探討這10種細(xì)菌是否存在水通道蛋白,為進(jìn)一步探討水通道蛋白的生理功能奠定基礎(chǔ)。 方法: 以10種細(xì)菌全序列基因組DNA為模板,用細(xì)菌水通道蛋白的保守氨基酸序列設(shè)計(jì)細(xì)菌水通道蛋白的簡(jiǎn)并PCR通用引物,進(jìn)行簡(jiǎn)并PCR擴(kuò)增,將得到的目的片段進(jìn)行PCR產(chǎn)物純化、測(cè)序;BLAST比較基因序列,判定是否存在細(xì)菌水通道蛋白。 結(jié)果: 簡(jiǎn)并PCR產(chǎn)物純化、測(cè)序后在大腸桿菌中發(fā)現(xiàn)410bp大小序列,BLAST證實(shí)為水通道蛋白Glp-F。新發(fā)現(xiàn)在痢疾志賀菌中存在370bp大小序列,BLAST證實(shí)為水通道蛋白Glp-F。在其余8種細(xì)菌中未發(fā)現(xiàn)類似水通道蛋白的DNA。 結(jié)論: 本試驗(yàn)證實(shí)簡(jiǎn)并PCR能夠利用已知的水通道蛋白保守氨基酸序列擴(kuò)增未知的DNA序列。探索到大腸桿菌存在水通道蛋白Glp-F,與國(guó)外報(bào)道一致,表明簡(jiǎn)并PCR能夠探索細(xì)菌水通道蛋白的存在;新發(fā)現(xiàn)痢疾志賀菌中存在細(xì)菌水通道蛋白Glp-F,而國(guó)外報(bào)道在福氏志賀菌中存在細(xì)菌水通道蛋白Glp-F,推測(cè)志賀菌各群中可能都存在水通道蛋白;本試驗(yàn)的發(fā)現(xiàn)為探索水通道蛋白Glp-F生理作用奠定了基礎(chǔ)。有8種細(xì)菌經(jīng)簡(jiǎn)并PCR擴(kuò)增未得到目的條帶,表明水通道蛋白在細(xì)菌種群之間的分布存在不均一性。 第二部分:高滲透壓誘導(dǎo)水通道蛋白Glp-F在痢疾志賀菌內(nèi)表達(dá) 目的: 用滲透壓不同的液體培養(yǎng)基和含有Hg2+(Glp-F選擇性抑制劑)的不同滲透壓培養(yǎng)基對(duì)痢疾志賀菌進(jìn)行培養(yǎng),誘導(dǎo)菌體內(nèi)Glp-F的表達(dá),以探討Glp-F蛋白在菌體內(nèi)的生理功能。 方法: 1.配置不同滲透壓培養(yǎng)基:普通牛肉浸液培養(yǎng)基;用無菌水稀釋為含1/3、1/2牛肉浸液的培養(yǎng)基;加入NaCl使?jié)B透濃度達(dá)到125mM、250mM、500mM、750mM培養(yǎng)基(N-原、N-1/3、N-1/2、N-125mM、N-250mM、N-500mM、N-750mM培養(yǎng)基)。 2.配置含有Hg2+抑制劑的不同滲透壓培養(yǎng)基:在上述培養(yǎng)基中分別加入Glp-F的功能抑制劑HgCl22mg得到H-原、H-1/3、H-1/2、H-125mM、H-250mM、H-500mM、H-750mM培養(yǎng)基。 3.在上述各種培養(yǎng)基中接種痢疾志賀菌,分別于24,48,72,96小時(shí)培養(yǎng)點(diǎn)上觀察細(xì)菌的生長(zhǎng)情況,用紫外分光光度計(jì)測(cè)定其OD600值以評(píng)價(jià)細(xì)菌的生長(zhǎng)情況。 4.用RT-PCR擴(kuò)增Glp-F的mRNA,瓊脂糖凝膠電泳、染色、成像分析,比較在不同環(huán)境條件下生長(zhǎng)的痢疾志賀菌Glp-F的mRNA轉(zhuǎn)錄量。 結(jié)果: 1.低滲透壓環(huán)境中培養(yǎng)的痢疾志賀菌在各個(gè)觀測(cè)點(diǎn)的生長(zhǎng)無明顯差異。 2.在N-培養(yǎng)基和H-培養(yǎng)基中生長(zhǎng)情況出現(xiàn)顯著不同:在H-培養(yǎng)基中細(xì)菌生長(zhǎng)速度明顯低于在N-培養(yǎng)基中生長(zhǎng)速度,特別是在125mM、250mM濃度培養(yǎng)基中變化最大。 3. RT-PCR顯示痢疾志賀菌Glp-F的mRNA含量在普通培養(yǎng)基、稀釋1/3、1/2水的牛肉浸液培養(yǎng)基、N-500mM/750Mm、H- N-500mM/750Mm培養(yǎng)基中沒有明顯差異;在N-125mM /250mM培養(yǎng)基和H-125mM /250mM中含量明顯增多,在H-125mM /250mM培養(yǎng)基中,細(xì)菌mRNA轉(zhuǎn)錄量大于在N-125mM/250mM培養(yǎng)基中的轉(zhuǎn)錄量。 結(jié)論: Glp-F是細(xì)菌特有的水通道蛋白,探討Glp-F的生理功能對(duì)研究細(xì)菌的水通蛋白有重要意義,國(guó)內(nèi)尚未見報(bào)道。本試驗(yàn)的結(jié)果可以得到以下結(jié)論: 1.表達(dá)Glp-F蛋白的痢疾志賀菌在低滲透壓下生長(zhǎng)繁殖速度沒有明顯變化。 2.在高滲透壓環(huán)境中,痢疾志賀菌的生長(zhǎng)繁殖速度隨著Glp-F蛋白功能的喪失而降低。 3. Glp-F的mRNA的在生長(zhǎng)期內(nèi)有不同的轉(zhuǎn)錄,對(duì)數(shù)生長(zhǎng)期內(nèi)尤為明顯,與國(guó)外報(bào)道相似。 4.滲透壓的升高能夠誘導(dǎo)Glp-F的表達(dá),尤見于N-125mM/250mM和H-125mM /250mM培養(yǎng)基中,并且各種滲透壓培養(yǎng)基中水通道蛋白mRNA轉(zhuǎn)錄量依次為H-250mMH-125mMN-250mMN-125mM原始培養(yǎng)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R378.25

【共引文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 李琦;紅色紅曲菌交替氧化酶基因Mraox1克隆與功能初步研究[D];華中農(nóng)業(yè)大學(xué);2010年

2 張倩;鵝源草酸青霉產(chǎn)纖維素酶條件優(yōu)化及CBHⅠ基因克隆[D];青島農(nóng)業(yè)大學(xué);2009年

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本文編號(hào)：2083549