本文選題：紅平紅球菌 + 非可培養(yǎng)�。� 參考：《蘭州理工大學(xué)》2017年碩士論文

【摘要】：細(xì)菌活的非可培養(yǎng)(VBNC)狀態(tài)是細(xì)菌在不良環(huán)境下形成的一種休眠方式。在所在環(huán)境改善時(shí)非可培養(yǎng)細(xì)胞復(fù)蘇為可培養(yǎng)狀態(tài),但有關(guān)細(xì)菌復(fù)蘇機(jī)制并不明確。藤黃微球菌(Micrococcus luteus)細(xì)胞復(fù)蘇促進(jìn)因子(Resuscitation promoting factor,Rpf)首次被報(bào)道能促使休眠菌狀態(tài)細(xì)胞復(fù)蘇,并刺激正常細(xì)胞生長(zhǎng)�，F(xiàn)已發(fā)現(xiàn)復(fù)蘇促進(jìn)因子(Rpf)廣泛存在于高G+C含量革蘭氏陽(yáng)性細(xì)菌中,與細(xì)菌生長(zhǎng)及抵御不良環(huán)境有關(guān)。不同種類細(xì)菌的Rpf種類和數(shù)量有一定差異。紅球菌屬的細(xì)菌廣泛分布于巖石、土壤、地下水等自然環(huán)境。多數(shù)種類在修復(fù)石油污染土壤、污水處理等方面有有廣闊應(yīng)用前景。紅平紅球菌(Rhodococcus erythropolis)KB1分離自石油污染土壤,具有高效石油降解能力和環(huán)境適應(yīng)性。為探索Rpf在紅平紅球菌適應(yīng)環(huán)境中的作用,本文進(jìn)行了紅平紅球菌KB1 Rpf基因克隆、基因結(jié)構(gòu)分析、表達(dá)及生物學(xué)性質(zhì)研究,具體內(nèi)容如下:設(shè)計(jì)了復(fù)蘇促進(jìn)因子基因特異引物,從紅平紅球菌KB1基因組DNA擴(kuò)增出4種rpf基因,分別為564、1125、1128和555 bp,編碼187、353、375和184個(gè)氨基酸殘基的蛋白質(zhì)。序列分析發(fā)現(xiàn)4種rpf基因分別與藤黃微球菌rpf基因,結(jié)核分枝桿菌(Mycobacterium tuberculosis)rpf B、rpf C及鏈霉菌(Streptomyces coelicolor)rpf B有較高相似性。4個(gè)rpf基因編碼的蛋白都有一個(gè)類似藤黃微球菌細(xì)胞復(fù)蘇促進(jìn)因子的Rpf域,含有1個(gè)保守谷氨酸殘基,由大約70個(gè)氨基酸組成。其中Rpf-1與結(jié)核分枝桿菌、鏈霉菌Rpf C相似,只含1個(gè)Rpf結(jié)構(gòu)域,Rpf-2與結(jié)核分枝桿菌Rpf B相似,含有1個(gè)Rpf域、1個(gè)G5域和2個(gè)DUF348,Rpf-3與鏈霉菌Rpf B相似,含有1個(gè)Rpf域、G5域和3個(gè)DUF348,Rpf-4與藤黃微球菌Rpf相似,含1個(gè)Rpf域和1個(gè)Lys M域。Rpf-2、Rpf-3含信號(hào)肽序列,Rpf-1、Rpf-4不含信號(hào)肽。Rpf-1、Rpf-2、Rpf-3和Rpf-4均為親水性蛋白,含有多個(gè)絲氨酸、蘇氨酸和酪氨酸位點(diǎn),沒(méi)有組氨酸位點(diǎn)。設(shè)計(jì)特異表達(dá)引物,將紅平紅球菌KB1 rpf-1基因克隆于原核表達(dá)載體p ET-32a(+),在大腸桿菌BL21中高效表達(dá),用Ni瓊脂糖親和層析柱純化重組蛋白,SDS-PAGE電泳分析表明純化蛋白分子量為36k Da。用人工合成底物4-Methylumbelliferyl-β-D-N-N’-N’-triacetyl chitotriose檢測(cè)發(fā)現(xiàn)rpf-1具有溶菌酶活性。溶菌酶比活力為2.07 U/mg。以偶氮酪蛋白為底物測(cè)定了純化蛋白rpf-1的蛋白酶活性,發(fā)現(xiàn)純化蛋白能水解偶氮酪蛋白,蛋白水解酶比活力為235 U/mg。將純化的重組蛋白R(shí)pf-1添加到不同狀態(tài)紅平紅球菌細(xì)胞培養(yǎng)物中,發(fā)現(xiàn)其對(duì)正常生長(zhǎng)、VBNC狀態(tài)及-80℃低溫保存的細(xì)胞具有明顯復(fù)蘇或促生長(zhǎng)作用。添加10%的Rpf-1使低溫保存紅平紅球菌生長(zhǎng)速度增長(zhǎng)4.8-6倍。添加Rpf-1使正常生長(zhǎng)的細(xì)菌細(xì)胞在12 h時(shí)入對(duì)數(shù)期,48 h細(xì)菌數(shù)量達(dá)最大,是對(duì)照組細(xì)菌生長(zhǎng)量的3倍。添加Rpf-1重組蛋白使VBNC狀態(tài)的紅平紅球菌復(fù)蘇量增加,48 h后細(xì)菌生長(zhǎng)量是空白對(duì)照組的1.44倍。
[Abstract]:The non-culturable VBNCstate of bacteria is a dormant way for bacteria to form in bad environment. The resuscitation of non-culturable cells was culturable when the environment was improved, but the mechanism of bacterial resuscitation was not clear. It was reported for the first time that Micrococcus luteus could promote the recovery of dormancy cells and stimulate the growth of normal cells. It has been found that the resuscitation promoter rpf) is widely found in Gram-positive bacteria with high G C content, which is related to bacterial growth and resistance to adverse environment. The species and quantity of RPF of different bacteria are different to some extent. Rhodococcus bacteria are widely distributed in rock, soil, groundwater and other natural environment. Most species have broad application prospects in oil contaminated soil remediation, sewage treatment and so on. Rhodococcus erythropolisus KB1 was isolated from petroleum contaminated soil and has high oil degradation ability and environmental adaptability. In order to explore the role of RPF in the adaptive environment of Rhodococcus hongpingensis, the gene cloning, gene structure analysis, expression and biological properties of KB1 Rpf gene were studied. The main contents were as follows: a specific primer for resuscitation promoting factor gene was designed. Four rpf genes encoding 187353375 and 184 amino acid residues were amplified from the KB1 genome of Rhodococcus rubrum. Sequence analysis revealed that four rpf genes were associated with rpf gene of Micrococcus lutei. Mycobacterium tuberculosis)rpf rpfC and Streptomyces coelicolor)rpf B have high similarity. The four rpf gene encoded proteins have a RPF domain similar to micrococcus luteum resuscitation factor, and contain a conserved glutamate residue. It consists of about 70 amino acids. Among them, Rpf-1 is similar to Mycobacterium tuberculosis, StrepfC, only one RPF domain Rpf-2 is similar to Mycobacterium tuberculosis RpfB, and one RPF domain, one G5 domain and two DUF348Rpf-3 are similar to Strepfella rpf-3. The Rpf-4 containing one RPF domain G5 and three DUF348C Rpf-4 is similar to Rpf of Micrococcus luteum, and contains one RPF domain and one Lys M domain. Rpf-2N Rpf-3 contains no signal peptide. Rpf-1Rpf-2 rpf-3 and Rpf-4 are hydrophilic proteins, and contain multiple serine, threonine and tyrosine sites, and Rpf-4 have multiple serine, threonine and tyrosine sites, and Rpf-1, Rpf-2, Rpf-3 and Rpf-4 are hydrophilic proteins with multiple serine, threonine and tyrosine sites. There is no histidine site. A specific expression primer was designed and cloned into the prokaryotic expression vector pET-32a (pET-32a). The recombinant protein was purified by Ni agarose affinity chromatography. The molecular weight of the purified protein was 36kDa. The synthetic substrates 4-Methylumbelliferyl- 尾 -D-N-Na-Na-N-triacetyl chitotriose showed that rpf-1 had lysozyme activity. The specific activity of lysozyme was 2.07 U / mg. The protease activity of purified protein rpf-1 was determined by using azo casein as substrate. It was found that the purified protein could hydrolyze azo casein and the specific activity of protein hydrolase was 235U / mg. The purified recombinant protein Rpf-1 was added to different cultures of Rhodococcus erythropoides. It was found that Rpf-1 could significantly resuscitate or promote the growth of normal growth cells in VBNC state and cryopreserved at -80 鈩，

本文編號(hào)：2025899