【作者】 郑会明；

【导师】 朱军；

【作者基本信息】 南京农业大学 ， 微生物学， 2013， 博士

【摘要】 菜豆根瘤菌可以在四季豆根部形成可以固氮的瘤体,目前对菜豆根瘤菌中群体感应的研究零星而且分散。目前仅报道了CFN42菌株的tra系统可以调节质粒的接合转移。在另一株菜豆根瘤菌CNPAF512中,根瘤菌可以产生AHLs结构物质,但具体的合成酶基因尚不明确。本论文以菜豆根瘤菌CFN42为研究对象,鉴别和分析了其中典型的三套群体感应系统,并发现该系统调节了根瘤菌中胞外多糖的产生和共生的过程。在线分析R.etli基因组序列信息,获得了三种luxR/luxI型群体感应基因单元,分别是cinRI, raiRI和traRI。cinRI位于染色体基因组中,raiRI位于质粒p42f中,traRI则位于可以自我发生质粒转移的质粒p42a中。克隆所研究的luxI、luxR基因两端侧翼序列至含sacB基因的自杀性质粒载体pEx18Gm中,接合至R.etli中,运用抗生素抗性和蔗糖致死筛选两次同源交换事件,由此获得了一系列luxI、luxR基因的单基因,双基因和三基因框内缺失菌株。对R.etli野生型菌株和luxl单基因缺失株的培养上清液进行乙酸乙酯抽提浓缩,利用电喷雾电离质谱(ESI-MS/MS)和TLC结合的方法分析了R.etli菌株产生的AHLs分子,初步确定Rail负责合成产生3-OH-C8-HSL, TraI负责合成3-oxo-C8-HSL和C8-HSL, CinI负责合成3-OH-C10-HSL。对所有的luxI、luxR缺失株产生AHLs分子的活性进行了测定,发现cinI.及cinR基因的缺失使R.etli基本丧失了产生AHLs分子的活性,结合本实验室相关研究数据(cin系统的缺失导致了raiI/R, traI/R启动子转录表达均失去活性),推测在R.etli中cin系统可能处于群体感应调控系统的上游,正向调控根瘤菌体内其他存在的群体感应基因,这在Rhizobium leguminosarum中有类似报道。从AHLs产生活性来看,traI/traR基因的缺失对R.etli AHLs产生活性影响较大,而raiI/raiR基因的缺失则并不明显改变R.etli活性的高低。推测其可能原因,一是.Tral/R在R.etli中酶活较高,合成了占根瘤菌体内绝大部分的AHLs, Rail/R活性较低,产生的AHLs比重较低。另一可能原因则在于所使用的AHLs的检测指示菌JZA1,该菌株是由一株缺失了Tral基因的Agrobacterium tumefaciens构建得来,并且对不同种类的AHLs分子检测实验结果也表明该菌株对AHLs的敏感性存在一些偏好。将luxI、luxR全基因片断克隆至Plac表达载体pYC12中,接合至R.etli相关缺失株。在luxI或luxR单基因缺失株中,raiRI, traRI, cinI均可以大幅增加原有缺失株AHLs的活性,这表明这三类群体感应基因确实负责产生合成了相应活性的AHLs分子。但cinR基因的表达并不能回补cinR缺失株AHLs分子产量的缺陷。在luxI和IuxR三基因缺失株的回补实验中则表明,cinIR系统所产生的AHLs分子占根瘤菌所有AHLs的比重极低。本文进一步研究了LuxR及LuxI蛋白对根瘤菌生理功能的影响,通常在群体感应调节系统中,LuxR蛋白可以结合在一些基因的启动子区域影响基因的表达进而调节生理功能。结果发现cin系统基因的缺失会降低根瘤菌胞外多糖产量和生物膜的形成,通过对可能的与根瘤菌中胞外多糖合成相关基因的启动子转录水平的分析,初步推测cin系统的存在降低了转录抑制子RosF蛋白基因的表达水平,进而促进了多糖相关基因的表达。通过菜豆结瘤实验检测了luxI、luxR单基因和三基因缺失株在菜豆植株根部共生的能力,试验结果表明所有的群体感应缺失株在最早结瘤时间方面无明显差异,在结瘤数量上cinR缺失株明显少于其他菌株,同时cinR缺失株所形成瘤体中大型粉红瘤所占比例也较高,而raiR缺失株所形成瘤体中大型粉红瘤所占比例则偏低。对固氮酶活力测定表明,cinR, raiR, tral突变株的固氮能力都较其他处理偏低。以上结果说明了在菜豆根瘤菌CFN42菌株中群体感应调控系统可以参与调节根瘤菌与植物的共生过程,尤其是.cinR基因的影响更为明显。本文还研究分析了luxR基因缺失株的竞争结瘤能力。结果表明,将luxR缺失株与野生型菌株共同接种具有结瘤优势,体现在结瘤数有所增加,瘤重显著增加。在108cell/mL接种量,与野生型菌株1：1混合接种下,luxR缺失株均一致表现出瘤体高占有率的现象。对cinR缺失株进一步研究,在不同接种量下cinR基因的缺失均表现出高结瘤竞争性,但当调整接种至cinR:WT=0.1,0.01时,cinR竞争性优势不再明显直至失去竞争能力。这表明在根瘤菌结瘤过程中,外源环境中的细胞数量与侵染的成功性直接正相关,其固有的竞争优势会随细胞密度的降低也不断的被减弱直至消失。综合以上,初步得出以CinR为代表的LuxR类蛋白基因的缺失,更有利于结瘤能力的增强,在R.etli中,群体感应的丧失也许更有利于根瘤菌的入侵和在根瘤中的相关生理行为。更多还原

【Abstract】 R.etli forms nitrogen-fixing nodules on the roots of of common bean, quorum sensing in R. etli is fragmentary but seems as complex as that of R. leguminosarum bv.viciae which had been best characterized. Till now, only tra genes involved in self-conjugative plasmid transfer was detailed described in R.etli CFN42. In another R.etli strain, CNPAF512, raiRI and cinRI had been reported, but the structure of these AHLs moleculses is still unkonwn but that cinRI for Hydroxylated long chain, raiRI for Short chain AHLs synthesis. In this work we identified the structures of all AHLs produced by three different synthase respectively, and show that these quorum sensing gens are involved in exopolysacchrides prodtion and symbiosis interation.Search for the genome sequence of R. etli CFN42 was performed, found other two genetic LuxR/I homologues indenpdent with Tra, designated CinR/I and RaiR/I, respective-ly locate on R.elti choromose and plasmid p42f. A serises of single,double, triple luxI/luxR in-frame deletion were constructed by overlapping PCR of flanking regions and cloned into the sacB suicide vector pEx18Gm to select for double-crossover events.To further study the roles of QS system of R. etli, the structure of AHLs molecules produced three LuxI synthases were analyzed in detail. The electrospray ionization mass spectrometry (ESI MS/MS) anyalisis conbined by thin-layer-chromography revealed that, Four AHL molecules were synthased by R.etli, Rail produced 3-OH-C8-HSL, Tral produced two components C8-HSL and3-oxo-C8-HSL, while CinI synthased long-chain AHLs:3-OH-C10-HSL. To investigate the functions of the three LuxI/R regulartory gene, we examined AHL prodution in a series of single, double, and triple mutants with in-frame deletions of QS genes. Mutation of single cinl or cinR surprisingly resulted in dramatic decrease of AHL accumulation, remaining about 1/100 AHL activity of that produced by wild type strain at early-stationary phase, this indicate that cinl and cinR may suite at the top of the QS system to regulate rai and tra QS genes.Mutations of rail and raiR did not significantly alter the AHLs activities observed in the parent stain. The loss of tral greatly reduced the production of putative AHLs detected, even changed the pattern of AHL production curve. Theses resutls imply that in R.etli, enzyme activity of Tral/R may be higher than RaiI/R, or for the AHLs detector, Agrobacterium tumefaciens, which has a bias for 3-oxo-C8-HSL which produced by TraI homologue.Plac-expressed luxl and luxR genes were introduced into responding QS mutants to determine AHLs activities produced by these complementary strains. All luxI or luxR type gene except cinR could restore AHLs production from luxI/luxR single mutant. CinI produced very lower AHLs activity in luxl triple mutant indicate that CinI synthase may was a lower-expressed enzyme in R.etli.In a QS system, active LuxR often act as a regulator by binding DNA region of specific target genes. The LuxR mutant was further investigated for phenotype defect caused by QS. Mutation of cinR or cinl gene lead to decreased EPS prodution and weaker biofilm formation in R.etli.The effect caused by cin QS system may mediated by transcriptional repressor RosR which could regulate genes of diverse function, including those involved in polysaccharide production and in carbohydrate metabolism.Symbosis phenotype of luxI/luxR single and triple mutants were investigated and revaled that no significane difference was observed in all QS mutans in first nodule apperance. cinR mutant showed significiant less noduled number but formed much more pink and big nodules than other treatments.RaiR formed less number of big pink nodules. cinR,raiR,traI also showed lower nitrogen fixting activities. The observation indicated that QS may play some roles in rhizobia-plant interation.We further investigated the competitiveness of three luxR mutant. All co-inoculation with WT and luxR could form more nodules and lead to increased dry nodule weight. When coinoculated with WT at a ratio of 1:1 in cell density of 108/ml, all luxR mutants showed high competitiveness. When inoculated with WT iat a ratio of 0.1, cinR showd same nodule capacity with WT, but in ratio of 0.01, cinR will lose infection ability. These results indicate that the strong competive ability of cinR could be decreased by cell number disadvantage. Above all, loss of LuxR, especially CinR, may have a advantage for nodulation in R.etli, to convenient host infection or other physical behavior.更多还原