本文選題：固氮施氏假單胞菌A1501 + 第二信使　； 參考：《安徽農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：環(huán)二鳥苷酸(c-di-GMP)是一種廣泛存在于細(xì)菌中的第二信使,參與調(diào)節(jié)多種生理功能,包括細(xì)胞分化、信號(hào)傳遞、生物膜形成、致病因子產(chǎn)生以及群體感應(yīng)系統(tǒng)的調(diào)控等。c-di-GMP由兩個(gè)GTP分子經(jīng)環(huán)化酶(DGCs)合成,而被磷酸二酯酶(PDEs)降解。環(huán)化酶(GTPs)具有保守的GGDEF結(jié)構(gòu)域,磷酸二酯酶(PDEs)具有保守的EAL結(jié)構(gòu)域。在銅綠假單胞菌中sadC為c-di-GMP合成的關(guān)鍵基因,而bifA基因?yàn)閏-di-GMP的降解基因,這兩個(gè)基因分別參與了胞內(nèi)c-di-GMP的濃度調(diào)節(jié),進(jìn)而影響菌體的運(yùn)動(dòng)、生物膜形成等表型。施氏假單胞菌(Pseudomonas stutzei)A1501是一株根際聯(lián)合固氮菌,c-di-GMP在該菌中的合成、降解機(jī)制還未得到鑒定。為研究A1501菌c-di-GMP的合成和降解途徑及其對(duì)菌株的影響,本研究將A1501中與銅綠假單胞菌sadC和bifA高度同源的兩個(gè)功能基因作為研究對(duì)象。通過構(gòu)建sadC和bifA功能缺失突變株,測(cè)定突變株胞內(nèi)的c-di-GMP濃度,進(jìn)而分析c-di-GMP水平的變化對(duì)菌體運(yùn)動(dòng)、生物膜形成等生理功能的影響。取得的主要研究結(jié)果如下:1、基因組分析表明,A1501中含有編碼GGDEF結(jié)構(gòu)域蛋白的基因共有33個(gè),含有編碼EAL結(jié)構(gòu)域蛋白的基因有17個(gè),其中編號(hào)為PST_3133的基因編碼氨基酸序列中含有一個(gè)GGDEF結(jié)構(gòu)域和一個(gè)EAL結(jié)構(gòu)域,與銅綠假單胞菌BifA氨基酸序列同源性高達(dá)74%,將其命名為bif A。編號(hào)為PST_1148的基因編碼包含一個(gè)GGDEF結(jié)構(gòu)域的蛋白,與銅綠假單胞菌中已報(bào)道的c-di-GMP環(huán)化酶SadC同源性達(dá)到62%,將其命名為sadC基因。采用三親結(jié)合的方法分別構(gòu)建了bifA、sadC功能缺失突變株,以及sadC過表達(dá)株和突變株bifA的功能回補(bǔ)株進(jìn)行后續(xù)研究。2、對(duì)sadC突變株的胞內(nèi)c-di-GMP含量及表型進(jìn)行了測(cè)定,結(jié)果表明sadC的功能缺失對(duì)菌體胞內(nèi)c-di-GMP含量與野生型相比沒有明顯差異,而且對(duì)菌株的生物膜形成能力以及固氮能力沒有顯著影響,但是菌體運(yùn)動(dòng)能力下降;透射電鏡觀察結(jié)果表明,sadC突變株與野生型的鞭毛形態(tài)沒有明顯差異。對(duì)sadC過表達(dá)菌株的生物膜形成能力進(jìn)行測(cè)定,結(jié)果表明sadC過表達(dá)菌株的生物膜形成能力比野生型增強(qiáng)約2倍以上。通過測(cè)定sadC過表達(dá)株胞內(nèi)c-di-GMP含量發(fā)現(xiàn)sadC過表達(dá)株胞內(nèi)c-di-GMP含量與野生型相比,提高約140%,說明sadC參與了A1501菌體內(nèi)的c-di-GMP合成途徑。3、測(cè)定bif A突變株的胞內(nèi)c-di-GMP含量,結(jié)果發(fā)現(xiàn)與野生型相比bifA突變株胞內(nèi)c-di-GMP含量增高約2倍以上;測(cè)定了bifA突變株的表型,結(jié)果顯示,與野生型相比,bifA突變株的生物膜形成能力增強(qiáng)約2倍,而bifA突變株的功能回補(bǔ)株生物膜形成能力與野生型類似。由此推測(cè),A1501菌中bifA基因可能是c-di-GMP的降解基因,該基因的功能缺失造成了菌體內(nèi)c-di-GMP的積累,進(jìn)而影響了菌體生物膜合成。表型測(cè)定也發(fā)現(xiàn)bif A的突變降低了菌體的運(yùn)動(dòng)能力。4、通過qRT-PCR分析了bifA基因在菌體不同生長(zhǎng)期的表達(dá)量,結(jié)果表明,bifA在野生型A1501的生長(zhǎng)初期有較高的表達(dá)量,而在指數(shù)期及平臺(tái)期表達(dá)量下降,暗示著bifA的表達(dá)水平可能與菌體濃度相關(guān)。通過添加不同濃度(0、10、20、40μM)的外源c-di-GMP考察對(duì)野生型生物膜形成的影響,結(jié)果發(fā)現(xiàn)外源c-di-GMP沒有影響菌體生物膜的形成,推測(cè)外源c-di-GMP無法轉(zhuǎn)運(yùn)至胞內(nèi)。以上結(jié)果表明,在固氮施氏假單胞菌A1501中,c-di-GMP作為細(xì)胞重要的第二信使,參與調(diào)控菌體的生物膜、運(yùn)動(dòng)性等重要生理過程。
[Abstract]:Cyclic diguanosine (c-di-GMP) is a second messenger widely used in bacteria. It participates in regulating a variety of physiological functions, including cell differentiation, signaling, biofilm formation, pathogenicity factor production and the regulation of.C-di-GMP by the synthesis of two GTP molecules via cyclase (DGCs), and degrading by phosphodiesterase (PDEs) and cyclization. The enzyme (GTPs) has a conservative GGDEF domain, and phosphodiesterase (PDEs) has a conservative EAL domain. In Pseudomonas aeruginosa, sadC is the key gene for c-di-GMP synthesis, while the bifA gene is a c-di-GMP degradation gene. These two genes are involved in the concentration regulation of intracellular c-di-GMP, and then affect the movement of the bacteria and the formation of the biofilm. Pseudomonas stutzei A1501 is a combination of rhizosphere nitrogen fixing bacteria and c-di-GMP in the bacteria, and the mechanism of degradation has not been identified. In order to study the synthesis and degradation pathway of A1501 bacteria c-di-GMP and its effect on the strain, two functional genes in A1501 are highly homologous to the sadC and bifA of Pseudomonas aeruginosa in A1501. As a study object, by constructing sadC and bifA functional deletion mutants, the c-di-GMP concentration in the mutant cell was determined, and then the effects of the changes of c-di-GMP level on the physiological function of the mycelium movement and biofilm formation were analyzed. The main results obtained are as follows: 1, the genome analysis shows that the A1501 contains the base of the GGDEF domain protein. A total of 33 genes contain 17 genes encoding EAL domain proteins, including a GGDEF domain and a EAL domain in the sequence of encoded amino acids numbered PST_3133. The homology of the BifA amino acid sequence of Pseudomonas aeruginosa is up to 74%. The gene encoding a BIF A. coded as PST_1148 contains a GGDEF knot. The homology of c-di-GMP cyclase SadC, which has been reported in Pseudomonas aeruginosa, is 62%. It is named sadC gene. BifA, sadC functional deletion mutant, sadC overexpression strain and functional bifA of mutant strain of bifA are constructed by three affinity methods, respectively, for the follow-up study of.2, and the intracellular c-di-G of the sadC mutant strain. The content and phenotypes of MP were measured. The results showed that the functional deletion of sadC had no significant difference to the intracellular c-di-GMP content of the mycelium, but had no significant influence on the biofilm formation ability and nitrogen fixing ability of the strain, but the motion ability of the bacteria decreased. The results of transmission mirror observation showed that the sadC mutant and the wild type whip. The biofilm formation ability of sadC overexpressed strain was measured. The results showed that the biofilm formation ability of sadC overexpressed strain was about 2 times more than that of wild type. By measuring the intracellular c-di-GMP content of sadC overexpressed strain, the intracellular c-di-GMP content of sadC overexpressed strain increased by about 140% compared with that of wild type. The results showed that sadC participated in the c-di-GMP synthesis pathway.3 in A1501 bacteria and determined the intracellular c-di-GMP content of BIF A mutant strain. The results showed that the c-di-GMP content in the bifA mutant was about 2 times higher than that of the wild type, and the phenotype of the bifA mutant was measured. The results showed that the biofilm formation ability of bifA mutant strain increased by about 2 compared with the wild type. The biofilm formation ability of the bifA mutant strain was similar to that of the wild type. Thus, it is assumed that the bifA gene in A1501 bacteria may be a degrading gene of c-di-GMP. The deletion of the gene's function causes the accumulation of c-di-GMP in the bacteria, and then affects the synthesis of the bacterial biofilm. The mutation of the bif A has also found that the mutation of the bif A reduces the transport of the bacteria. Dynamic ability.4, the expression of bifA gene in different growth stages was analyzed by qRT-PCR. The results showed that bifA had higher expression in the early stage of growth of wild type A1501, while the expression level in the exponential phase and platform stage decreased, suggesting that the expression level of bifA may be related to the concentration of the bacteria. By adding different concentrations (0,10,20,40 mu M), the expression level of bifA was related. The effect of c-di-GMP on the formation of wild type biofilm was investigated. The results showed that exogenous c-di-GMP did not affect the formation of the bacterial biofilm. It was suggested that exogenous c-di-GMP could not be transported to the intracellular. The above results showed that in the A1501 of Pseudomonas sp., c-di-GMP was an important second messenger of the cell, and participated in the regulation of the biofilm of the bacteria and the weight of motion. Take a physiological process.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q93;Q78

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 史德青,趙金生,楊金榮,侯影飛,孔瑛;施氏假單胞菌對(duì)二苯并噻吩的降解[J];中國環(huán)境科學(xué);2004年06期

2 魏曉星;劉峰;簡(jiǎn)嘉;王瑞妍;陳國強(qiáng);;重組施氏假單胞菌利用非相關(guān)碳源生產(chǎn)3-羚基丁酸與3-羚基己酸共聚酯(英文)[J];Chinese Journal of Chemical Engineering;2013年09期

3 胡國元;張凱;袁軍;楊洋;雷夢(mèng)婕;章建國;;施氏假單胞菌菌株的脫氮特性[J];武漢工程大學(xué)學(xué)報(bào);2012年06期

4 李文明;楊文華;李海星;劉曉華;曹郁生;;施氏假單胞菌F4對(duì)黃曲霉毒素B_1的酶解作用及其降解產(chǎn)物的初步分析[J];食品與發(fā)酵工業(yè);2013年02期

5 侯影飛;孔瑛;郭寧;李春虎;;施氏假單胞菌UP-1降解二苯并噻吩的動(dòng)力學(xué)模型[J];石油學(xué)報(bào)(石油加工);2009年05期

6 尚立國;弓湃;戰(zhàn)崳華;鄧志平;燕永亮;;固氮施氏假單胞菌全局性調(diào)控因子RsmA的進(jìn)化分析及表達(dá)特性研究[J];中國農(nóng)業(yè)科技導(dǎo)報(bào);2014年03期

7 楊文華;劉曉華;李文明;李海星;曹郁生;;施氏假單胞菌F4產(chǎn)黃曲霉毒素B_1降解酶條件的優(yōu)化[J];食品科學(xué);2013年09期

8 王全偉;張海玲;孟慶英;徐香玲;李新玲;;施氏假單胞菌Na~+/H~+逆向轉(zhuǎn)運(yùn)蛋白基因nhaA的克隆與表達(dá)分析[J];中國農(nóng)學(xué)通報(bào);2010年15期

9 劉陳立,邵宗澤;海洋石油降解微生物的分離鑒定[J];海洋學(xué)報(bào)(中文版);2005年04期

10 ;[J];;年期

相關(guān)會(huì)議論文 前1條

1 楊智敏;林敏;燕永亮;;固氮施氏假單胞菌glnD基因突變株構(gòu)建及表型分析[A];第四屆全國微生物資源學(xué)術(shù)暨國家微生物資源平臺(tái)運(yùn)行服務(wù)研討會(huì)論文集[C];2012年

相關(guān)博士學(xué)位論文 前4條

1 史延華;農(nóng)藥降解菌施氏假單胞菌YC-YH1的分離鑒定及降解機(jī)理研究[D];中國農(nóng)業(yè)科學(xué)院;2015年

2 鄧斌;施氏假單胞菌SC221-M的反硝化特性及其調(diào)節(jié)草魚養(yǎng)殖水體水質(zhì)的研究[D];浙江大學(xué);2014年

3 張小平;枯草芽孢桿菌SC02和施氏假單胞菌F1M對(duì)草魚養(yǎng)殖水體水質(zhì)的影響及機(jī)理研究[D];浙江大學(xué);2014年

4 馬堯;固氮施氏假單胞菌非編碼RNA CrcZ和CrcY介導(dǎo)的碳代謝物抑制調(diào)控[D];中國農(nóng)業(yè)大學(xué);2015年

相關(guān)碩士學(xué)位論文 前10條

1 孫世英;納米鐵共存條件下施氏假單胞菌對(duì)BDE-47的吸附/降解的研究[D];華南理工大學(xué);2015年

2 王巍;固氮施氏假單胞菌RNA分子伴侶蛋白Hfq的功能鑒定及表達(dá)特性研究[D];華中農(nóng)業(yè)大學(xué);2015年

3 陸超;固氮施氏假單胞菌鐵吸收調(diào)節(jié)蛋白Fur參與鐵轉(zhuǎn)運(yùn)和固氮調(diào)控的研究[D];中國農(nóng)業(yè)科學(xué)院;2016年

4 李耘;固氮施氏假單胞菌RpoN蛋白調(diào)控硝酸鹽同化的作用機(jī)制[D];中國農(nóng)業(yè)科學(xué)院;2016年

5 柯琪;固氮施氏假單胞菌RNA分子伴侶蛋白Hfq與碳代謝抑制蛋白Crc在碳代謝途徑中的協(xié)同作用[D];西南科技大學(xué);2016年

6 盧佳思;固氮施氏假單胞菌環(huán)二鳥苷酸（c-di-GMP）代謝相關(guān)基因的功能鑒定[D];安徽農(nóng)業(yè)大學(xué);2016年

7 李琴;固氮施氏假單胞菌一般氮代謝調(diào)控蛋白NtrC的功能鑒定[D];安徽農(nóng)業(yè)大學(xué);2013年

8 趙靜;施氏假單胞菌N2降解酚類污染物的特性及機(jī)理研究[D];西安建筑科技大學(xué);2013年

9 尚立國;固氮施氏假單胞菌次級(jí)代謝抑制因子RsmA的功能研究[D];中國農(nóng)業(yè)科學(xué)院;2014年

10 李燕;固氮施氏假單胞菌一般脅迫反應(yīng)蛋白R(shí)poS的鑒定和作用機(jī)制研究[D];中國農(nóng)業(yè)科學(xué)院;2010年

，

本文編號(hào)：2106090