外源AHL对中慢生型天山根瘤菌群体感应系统作用的初步研究

通过将天山根瘤菌中的自体诱导物合成酶基因mrtI在转录水平融合肠lacZ构建了5'mrtI-lacZ融合表达菌株HMZ1.利用多株不同属的根瘤菌上清液对该菌株进行诱导,发现有3株豌豆根瘤菌和1株三叶草根瘤菌上清提取物能诱导mrtI基因的表达,且外源上清量越多,mrtI被诱导的程度越高,TLC的结果则显示它们可能有着相同的AI.该结果显示在相同功能的细菌间存在信号交流,为进一步研究群体感应调节在共生固氮上的作用提供了理论及实践依据.     

中慢生型天山根瘤菌中自体诱导物分子结构的鉴定

通过用乙酸乙脂分别对天山根瘤菌 CCBAU3306 培养物上清液中自体诱导物分子进行抽提、浓缩,并采用液质联用对自体诱导物分子进行结构分析,最终获得该根瘤菌所产生酰基高丝氨酸内酯类信号分子的结构,并采用纯品进行活性的比较,发现调控蛋白与纯品结合后具有生物活性,能与目的 DNA 片断结合,证明了该结构的准确性.     

自体诱导物合成酶基因的筛选及其功能分析

细菌在高细胞密度下通过产生一类化学信号分子(autoinducer,自体诱导物)感知细胞群体密度变化,从而对基因表达进行调控,这一现象被称为群体感应(quorum sensing).以中慢生型华癸根瘤菌 Mesorhizobium huakuii AS9 为出发菌株,利用转座子随机突变的方法获得了自体诱导物缺失突变株 YQ1,并克隆得到了其自体诱导物合成酶基因,命名为 mrhI;构建PmrhI-lacZ 转录融合表达质粒 pYQ1,通过同源重组的方法得到在华癸根瘤菌中 mrhI-lacZ 融合表达而且 mrhI 基因突变菌株YQ3.突变株 YQ3 的培养液上清不含有自体诱导物分子,通过 lacZ 报告基因的表达发现 mrhI 的表达受自体诱导物的调控,说明 mrhI 基因是 M. huakuii AS9 的群体感应体系中的自体诱导物合成酶基因.     

中慢生型天山根瘤菌CCBAU060A自体诱导物合酶基因的筛选及其在大肠杆菌中的表达

细菌在高细胞密度下可以产生化学信号分子调控细菌相关基因的表达,这种信号分子被称为自体诱导物(Autoinducer,AI)。采用检测菌株JZA1在中慢生型天山根瘤菌CCBAU060A(Mesorhizobium tianshanenseCCBAU060A)培养上清液中检测到了高活性的自体诱导物;利用转座子随机突变的方法获得了AI缺失突变株,并克隆得到了相关的自体诱导物合酶基因;将自体诱导物合酶基因在大肠杆菌中进行表达,对大肠杆菌重组菌株进行自体诱导物检测发现,该合酶基因在大肠杆菌中能够合成四种自体诱导物分子。     

百脉根根瘤菌自体诱导物合成酶基因mrlI2的初步研究

许多细菌均能产生一类叫做自体诱导物的信号分子,用于感知自身密度的变化,从而调控其生理行为,这种现象称为群体感应[1]。越来越多的研究表明,在根瘤菌中普遍存在着群体感应调控系统[2],并且它可能在根瘤菌与豆科植物共生结瘤固氮过程中起着重要作用。目前,在豌豆根瘤菌中共发现有4种群体感应系统(rai,rhi,cin和tra),其中cin系统位于上游,起总调控作用,tra系统控制着质粒的转移,rhi系统与结瘤数量有关,rai系统的功能还不清楚,它们共同组成了一个复杂的群体感应调控系统[3]。在苜蓿中华根瘤菌中,也发现群体感应系统与根瘤菌的生长和结瘤效率有关[4]。对中慢生型华癸根瘤菌和天山根瘤菌的研究     

中慢生型天山根瘤菌中MrtR蛋白的融合表达研究及鉴定

通过PCR扩增出中慢生型天山根瘤菌中群体感应调控蛋白MrtR的基因片段,克隆至表达载体pALEX中,从而构建了原核表达质粒pALEX-mrtR.将pALEX-mrtR转化至大肠杆菌BL2/DE3菌株感受态细胞中,经IPTG诱导,表达出58 kD的GST-MnR融合蛋白.用纯化好的GST-MrtR制备多克隆抗体.经Westem blot检测,该血清可与目的蛋白发生特异性反应,证明其具有良好的免疫原性.MrtR融合蛋白的成功表达及其多克隆抗体的制备为进一步研究MrtR蛋白的功能奠定了基础.     

糖类碳源对中慢生华癸根瘤菌群体感应基因mrhI/R表达的影响

克隆了中慢生华癸根瘤菌(Mesorhizobium huakuii)的mrh I和mrh R群体感应基因的启动子区域,至Lac Z翻译融合表达质粒载体p RA302中,经β-半乳糖苷酶活性检测发现,mrh I和mrh R的表达均受mrh I基因产物(自体诱导物)的正向调控。两种PTS糖类:葡萄糖和果糖的外源添加可以显著增加细菌的生长量,但却都同时抑制了mrh I和mrh R基因的表达,暗示在中慢生华癸根瘤菌中群体感应基因的表达可能受细菌中普遍存在的碳源代谢物阻遏现象的调节。     

紫云英根瘤菌种内不同菌株群体感应信号分子差异性和交互性分析

根瘤菌可以和豆科植物共生固氮,在很多根瘤菌中群体感应系统参与了结瘤固氮等相关生理过程的调节。本研究从野外不同生境紫云英宿主根部瘤体中分离了26株根瘤菌,体外定量检测了各菌株产生自体诱导物(AI)信号分子的能力,以及不同菌株AI激活中慢生华癸根瘤菌Mh菌株AHLs合成酶基因mrh I表达的情况,发现紫云英根瘤菌种内不同菌株间存在群体感应交互性。通过导入外源Att M水解酶观察菌株AI活性的变化,应用薄层层析法定性分析不同菌株的AI结构,初步揭示了不同紫云英根瘤菌产生AI特性的差异和多样性。     

慢生型大豆根瘤菌nfeC基因的定位

在前期工作中 ,从慢生型大豆根瘤菌菌株 GX2 0 1的基因文库中筛选到一个含 nfe C基因同源片段的重组质粒 p GXN2 0 1。在本工作中 ,将 nfe C基因同源片段定位在 4 kb Cla I Xba I片段上。对该片段的部分序列分析表明与已报道的 nfe C基因具有 95%的同源性     

慢生型大豆根瘤菌putA基因的功能分析

重组质粒pGX300上的putA基因是从慢生型大豆根瘤菌GX201中克隆到的编码脯氨酸脱氢酶的基因。通过Tn5gusA5定位诱变方法构建了putA基因的突变株GX20120,实验表明,GX20120的结瘤时间推迟,竞争结瘤能力降低。     

细菌胞外聚合物：基于土壤生态功能的视角

胞外聚合物（Extracellular Polymeric Substances,EPS）是微生物分泌到胞外环境的高分子量天然聚合物,其建立了生物膜结构和功能的完整性,是决定生物膜物理化学特性的关键组分。EPS是细菌生命的基础,为化学反应发生、养分捕获和抵抗环境胁迫提供了理想的环境。细菌分泌的EPS可以通过自身特性（如黏着性、吸湿性和络合性能等）实现土壤功能增益,如提高团聚体稳定性、增强保水持水能力,以及固持重金属等。本文梳理了细菌EPS的土壤生态功能,即充当细胞保护层、调节土壤生物响应、缓解土壤非生物胁迫和改善土壤整体功能,并探讨了土壤中细菌EPS未来研究的着眼点,以期呼吁更多学者关注细菌EPS及其在土壤生态系统中的功能。     

Surface and subsurface microbial biomass, community structure and metabolic activity as a function of soil depth and season

Microbial biomass, size and community structure along with an estimate of microbial activity and soil chemical parameters were determined at three depths in two soils (e.g. sandy loam Ultic Hapludalf and silt loam Mollic Hapludalf) replicated three times under one winter and summer season. Microbial biomass and community structure were estimated from phospholipid-PO₄ content and fatty acid methyl ester (FAME) measurements. Microbial activity and assimilative capacity were estimated using a ³H-acetate incorporation into phospholipids and by incubating the soil samples at the average winter and summer temperatures, 3 and 20 °C, respectively. We found that the size of the microbial biomass in both the surface and the subsurface soils was not significantly affected by the seasonal variation but activity increased by as much as 83% at the summer temperatures in the surface soil. We demonstrated using FAME analysis that for both soils seasonal changes in the subsurface microbial community occurred. These findings suggest that winter conditions will shift the population activity level in both the surface and subsurface systems and the biochemical structure of the community in the subsurface. In all cases, the inorganic chemical properties of the soil, as a function of season, remained constant. The greatly increased activity of microbial population at the higher temperature will favor the capacity of the system to utilize nutrients or organic materials that may enter soil. During low temperature seasons the capacity of either surface or subsurface soils to assimilate materials is generally diminished but the reduction reflects changes in metabolism and not a reduced biomass size.     

植物细菌天山根瘤菌调控基因mrtR在群体感应中的作用

在许多种类的细菌中均发现了luxR/luxI类型的群体感应调控系统,其中luxR是调节基因,对luxI合成自体诱导物(AI,autoinducer)起着正调控作用。通过建库筛选后,测序比对发现天山根瘤菌中存在mrtR/mrtI系统。通过基因敲除和转录水平基因表达对mrtR进行研究,发现mrtR严格调控着mrtI的表达,同时mrtR的表达也受到AI影响,这表明mrtR表达的调控是一种自调控,同时根毛吸附实验显示该基因对根瘤菌与宿主间相互作用密切相关。     

Biotoxic Effects of Herbicides on Growth,Nodulation, Nitrogenase Activity,and Seed Production in Chickpeas

Abstract

The effects of pre‐emergent (PRE) application of methabenzthiazuron (MBT), terbutryn, and linuron on plant vigor, number of nodules formed, nitrogenase activity (NA), chlorophyll contents, nitrogen (N) uptake, and seed yield were assessed in inoculated chickpeas, grown in sandy clay loam soil, in a controlled environment. The PRE application of MBT, terbutryn, and linuron at 2.5, 1.25 and 2 g a.i. kg^?1, respectively, had no significant adverse effect on plant vitality, yield, and NA of excised nodules in inoculated chickpea plants, but reduced these parameters at 7.5, 3.75, and 6 g a.i. kg^?1 of soil. Among the herbicides tested, linuron at each dose rates significantly (P≤0.05) decreased the grain yield, whereas terbutryn at 1.25 and 2.5 g a.i. kg^?1 improved the grain yield by 19.4% and 11%, respectively, relative to the nonherbicide control. While comparing the sum of mean values of toxicity of all dose rates of each herbicide on grain yield, the toxicity increased in the following order: control=linuron>methabenzthiazuron>terbutryn. Nodulation (nodule number per plant and their dry mass) and chlorophyll contents per plant decreased consistently with increased herbicide rates, except linuron, which at 2 g a.i. kg^?1 improved nodulation at both 45 and 90 days after seeding and corresponded with Mesorhizobium activity. The N content in grain and straw significantly decreased at the highest tested rates of each herbicide. Linuron at 2 and 4 g a.i. kg^?1 improved N content in straw, whereas at 4 g a.i. kg^?1 it significantly reduced N content in grain.     

Inoculation with Phosphate Solubilizing Mesorhizobium Strains Improves the Performance of Chickpea (Cicer aritenium L.) Under Phosphorus Deficiency

The use of phosphate-solubilising bacteria as inoculants increases plant phosphorus (P) uptake and thus crop yield. Strains from the genus Mesorhizobium are among the most powerful phosphate solubilizing microorganisms. In order to study efficiency in P uptake and N₂ fixation in chickpea (Cicer aritenium), forty-two rhizobia strains natively from Tunisian soils were studied in symbiosis with the chickpea variety “Béja1” which is frequently cultivated in Tunisia. Plants were inoculated separately with these strains under controlled conditions in perlite under two sources of P i.e. soluble (KH₂PO₄) and insoluble P (Ca₂HPO₄). At flowering stage, growth, nodulation, P uptake and N₂ fixation were assessed in all symbiotic combinations. The results showed that the S27 strain efficiently mobilized P into plants, observed as a significant increase of plant P content when insoluble P (Ca₂HPO₄) was supplied to the soil. This was associated with a significant increase in plant biomass, nodule number and N content under insoluble P conditions. Additionally, inoculation with the Mesorhizobium strain S27 significantly increased the root acid phosphatase activity under insoluble P. This study also shows significant correlations found between plant P content and acid phosphatase activity under low P conditions which may highlight the contribution of acid phosphatases in increasing P use efficiency. A field experiment also showed that most of the chickpea analyzed parameters were improved when plants inoculated with two selected rhizobia strains (S26 and S27) and supplied with P2O5. Overall, these findings postulate that rhizobial inoculation should not only be based on the effectiveness of strains regarding N fixation, but also to other traits such as P solubilisation potential.