【作者】 蔡韬；

【导师】 朱军；

【作者基本信息】 南京农业大学 ， 微生物学， 2010， 博士

【摘要】 中慢生型天山根瘤菌可以在其宿主甘草根部共生结瘤固氮,为植物提供生长所需的氮源。共生关系的建立需要根瘤菌和豆科植物之间复杂的信息交流,两者间的信息交流在豆科植物种子萌发时就已经开始了。种子萌发时产生大量的营养物质,包括糖类、氨基酸类的小分子物质,可以诱导土壤中细菌的趋化作用。根瘤菌在豆科植物根部形成侵入线并最终结瘤固氮的过程是目前研究的热点,根瘤菌中大量基因的表达或抑制是这一过程所必须的。但对于根瘤菌和豆科植物互作初期的研究却很少,特别是在根瘤菌吸附在根部之前的过程。本文旨在研究甘草在萌发时对天山根瘤菌基因表达的影响。在琼脂平板中添加不同浓度的甘草种子分泌物来模拟萌发时种子周围的化学环境。通过转座子插入的方法,筛选得到5株转座子插入可被甘草种子分泌稳定诱导的突变株(MsiA,B,C,D,E),其中MsiA可以被甘草种子分泌物诱导50倍。随机引物PCR(Arbitrary PCR)方法证明MsiA中转座子插入的基因属于LysE家族。甘草种子分泌物对MsiA的诱导作用需要MsiA上游基因msiR, MsiR属于LysR调控蛋白家族。对于甘草种子分泌物中的有效成分进行质普测定,发现能诱导MsiA基因表达的物质为刀豆氨酸。刀豆氨酸是精氨酸的类似物,可以竞争性地掺入氨基酸序列中,形成无功能蛋白,最终导致细菌的死亡,是一种抗菌物。精氨酸和赖氨酸可以诱导大肠杆菌(Escherichia coli)和谷氨酸棒杆菌(Corynebacterium glutamicum)中LysE基因的表达,但是不能诱导MsiA基因的表达。在体外酵母粉含量低于0.005%(W/V)的情况下,刀豆氨酸对msiA或msiR缺失突变株的毒性要比野生型高近一万倍。检测不同突变株胞内刀豆氨酸浓度发现msiA和msiR缺失突变株胞内刀豆氨酸的浓度要高于野生型,间接说明MsiA的功能是将胞内的刀豆氨酸运输到胞外,将胞内刀豆氨酸的浓度维持在对菌体无害的水平。根毛吸附实验发现msiA突变株在甘草根毛上的吸附量要比野生型低100倍。从甘草根部取得根际土,分离细菌并对其对刀豆氨酸的耐受性进行检测,发现甘草根际土壤菌群中耐受刀豆氨酸菌株数(NCR)与刀豆氨酸敏感菌株数(NcS)的比值(NCR/NCS比空白土样中的高八倍。这说明,甘草分泌的刀豆氨酸对其根际菌群具有一定的筛选作用。天山根瘤菌MsiAR系统和其宿主甘草种子萌发初期互作模型：在甘草萌发初期,种子和植株分泌的抗菌物质刀豆氨酸对根际菌群进行一次初步筛选,杀死植株周围可能的有害细菌；而MsiAR系统的存在使得天山根瘤菌可以在甘草根际选择性的环境下继续繁殖,成为优势菌群,为后来的结瘤共生提供有力条件。更多还原

【Abstract】 Mesorhizobium tianshanense is a nitrogen-fixing bacterium which can establish symbiotic associations with Glycyrrhiza uralensis. Complicated Signal communication are required for Establishment of symbiosis, this communication may occur as early as seed germination. During germination, seeds secret a lot of nutrient small molecular, including sugars, amino acid, which can induce chemotaxis of bacteria in soil.Formation of invasion thread, nodulation and nitrogen fixation are studied significantly, many genes are found necessary for these procedures. But there are few study on the early interaction between Rhizobium and their host plant, especially before attachment of bacteria on root hair. So my project was focused on how the compounds secreted during Glycyrrhiza uralensis seed germination affect M. tianshanense gene transcription profile, those genes whose transcription changed may play important roles in the early interaction between Rhizobium and plants.We put exude of Glycyrrhiza uralensis seed into agar plates to imitate the chemical surroundings of seed germination. Five genes(msiA,B,C,D,E) were identified to be induced by seed exude, by using a transposon containing a promoterless kanamycin resistant gene. msiA, which can be induced 50 times by seed exude, belongs to lysE family which encoding amino acid exporter protein.The induction of seed exude on msiA promoter needs the product of msiR, which located upstream of msiA, MsiR belongs to LysR regulation family. MS result showed that the functional compound in seed exude is canavanine, which is an analogue of arginine and can be misintegrated into polypepetide to form unfuntional protein, leading death of bacteria finally. Arginine and lysine, which can induce the expression of lysE in Escherichia coli and Corynebacterium glutamicum separately, cannot induce msiA expression.Canavanine was 104 times more toxic to msiA or msiR mutant than to wild type strain under culture condition where yeast extract was as low as 0.005%(W/V), also the intracellular canavanine concentration of msiA or msiR mutant was higher than wild type strain, suggesting that MsiA/R system may act as canavanine exporter to maintain intracellular canavanine to a nontoxic level. Root hair attachment experiment showed that the amount of msiA mutant attached to root hair is 100 times lower than wild type strain.We also separated stains from rhizosphere of blank soil or soil in which Glycyrrhiza uralensis were planted, tested the resistance of these strains and calculated the ratio(NCR/ NCS) of canavanine resistant strains(NCR) and canavanine sensitive strains(NCR) from different samples. We found that the ratio ratio(NCR/NCS) from plant soil is 8 times higher than from blank soil, suggesting that Glycyrrhiza uralensis may secret canavanine to change bacteria population in rhizosphere.Based on these data, we put forward the model for early interaction between M. tianshanense MsiA/R system and Glycyrrhiza uralensis:during germination, seed can secret canavanine as an antimicrobial to optimize the bacteria population in the rhizosphere, killing or repressing the potential hazard bacteria. But beneficial bacteria such as M. tianshanense can propagate to be the dominant population paving the way for further symbiosis, cause of MsiA/R system.更多还原