【作者】 郝秀丽；

【导师】 韦革宏；

【作者基本信息】 西北农林科技大学 ， 微生物学， 2013， 博士

【摘要】 根瘤菌-豆科植物共生体系在重金属污染土壤的生物修复中具有很大的应用前景，而揭示根瘤菌的重金属抗性机制及其在金属胁迫下和植物的共生关系是运用根瘤菌-豆科植物联合修复系统的基础和前提。本文通过全基因组测序分析、转座子突变等技术，对分离自甘肃铅锌矿区刺槐（Robinia pseudoacacia）根瘤中的两株重金属抗性细菌Agrobacterium tumefaciens CCNWGS0286及Mesorhizobium amorphae CCNWGS0123的Cu/Zn抗性机制、以及对Cu/Zn污染土壤的联合修复作用进行了研究。经测定，Cu²⁺对菌株CCNWGS0286和CCNWGS0123的最小抑制浓度（MIC）分别为2.8mM、2.5mM，而Zn²⁺对菌株的MIC分别为3.0mM、2.0mM。菌体对Zn²⁺的最适吸附条件及生物吸附特征的研究表明，在应用菌株CCNWGS0286和CCNWGS0123的活体细胞作为生物吸附剂时，对Zn²⁺生物吸附的最适条件为pH：5.0，生物吸附剂剂量：1.0g/L，初始金属浓度：100mg/L。多种动力学模型拟合结果表明，二级动力学模型能更加准确的描述菌株对Zn²⁺的吸附过程。Langmuir和Freundlich模型对菌体CCNWGS0286和CCNWGS0123吸附拟合效果不尽相同，但两菌株作为生物吸附剂对Zn²⁺的吸附均属于化学吸附。此外，两株菌体吸附剂均在pH为1.0时解析效率最高，达70%以上。结合傅里叶红外光谱、扫描电镜及能谱分析等方法，最终确定了-COOH、C=O、-NH和-C6H5等官能团主要参与了菌体吸附剂对Zn²⁺的吸附过程，而菌体可通过细胞形态变化和胞外分泌物的产生等途径增强菌体对Zn²⁺的抗性。A. tumefaciens CCNWGS0286（176）具有良好的重金属抗性及促植物生长能力。基于全基因组序列分析，发现并预测了A. tumefaciens176基因组中与锌抗性相关的基因及操纵子。通过转座子突变技术，建立了库容为7600的突变体文库，并从中筛选出9个锌敏感突变体。敏感性实验证明，多数突变体对Zn²⁺和Cd²⁺专一敏感、且敏感程度较高，此外，其中一株锌敏感突变体15-6IAA分泌能力显著下降。结合全基因组序列分析、转座子突变获得的插入基因以及锌抗性相关基因RT-PCR结果，最终确定在A.tumefaciens176中，P1b-type APTase ZntA-4200及MerR family转录调节因子ZntR1在维持细菌Zn和Cd抗性/稳态中起主要作用。此外，脂转运蛋白基因的缺失对菌体Zn²⁺、Cd²⁺抗性及IAA合成均有不同程度的影响，但其作用机制还不明确，可能与细菌的某些代谢活动相关。植物实验表明，A. tumefaciens176在Zn²⁺胁迫条件下仍能促进刺槐生长。通过菌株促植物生长因子测定及KEGG代谢途径分析，证明菌株能过量分泌吲哚乙酸（IAA），且IAA分泌量受Zn²⁺影响较小，因此推断能过量分泌IAA是A. tumefaciens176在Zn²⁺胁迫下促进植物生长最主要的原因。通过比较Zn²⁺胁迫条件下，野生菌176（ZnR，IAA+）、突变体12-2（ZnS，IAA+）、突变体15-6（ZnS， IAA-）以及参比菌株C58（ZnR，IAA-）对刺槐生长的促进作用，最终证明细菌金属抗性基因不能对抗或缓解金属对植物的毒害，植物生长激素才是污染条件下辅助植物存活、生长的主要因素。本研究全面分析了M. amorphae CCNWGS0123（186）全基因组序列特征，以及潜在的铜抗性相关基因及操纵子。同时结合转座子突变技术，构建了库容为10,000余株的突变体库，并筛选获得8个铜敏感性突变株。所有突变体Tn5插入基因主要分为三种类型：P-type ATPase、未知功能蛋白、及其他（GTP结合蛋白、核蛋白）。转座子突变基因及RT-PCR结果表明，CopA-6910和CusB共同决定M. amorphae186对铜的抗性。其中，CopA-6910主要负责将细胞质中的一价Cu排出到周质空间，而CusAB主要负责将周质空间内的一价Cu排出到细胞外，从而降低周质空间一价Cu的毒害。促植物生长因素测定结果表明，共生固氮能力是M. amorphae186在铜胁迫环境下辅助刺槐生长的主要因素。野生型菌株186及其铜敏感突变体3-42（copA::Tn5）和34-28（ccmX::Tn5）均可与宿主植物共生结瘤，但突变体34-28由于CcmX的缺失，其有效根瘤数量、固氮能力、促植物生长能力、及接种植物中Cu富集量均受到不同程度的影响。这说明CcmX的缺失，不仅影响菌株的铜抗性水平，同时还影响了菌株与宿主刺槐的共生结瘤过程，推测该基因可能与铜离子参与cbb3细胞色素c氧化酶系统中的催化过程有关。铜胁迫条件下植物实验表明，接种有186的刺槐，其全N含量、生物量以及Cu富集量比未接种的植物均有显著增加，且刺槐根中富集的Cu远远高于茎叶Cu富集量，进一步说明了将根瘤菌-刺槐共生体系用于修复矿区重金属污染环境的潜能。更多还原

【Abstract】 Due to the ability of nitrogen fixation by rhizobia, rhizobia-legumes symbiosis plays animportant and potential role in aiding phytoremediation of some metal contaminated soils. Toobtain the maximum benefits from legumes assisted by rhizobia for phytoremediation ofmetals, it is critical to have a good understanding of the metal resistant machenism of rhizobiaand the symbiotic plant-rhizobia relationship with metals. In this study, analysis of thegenome sequence and of transcription via RT-PCR combined with transposon genedisruptions revealed the copper or zinc resistance of Agrobacterium tumefaciensCCNWGS0286and Mesorhizobium amorphae CCNWGS0123isolated from the nodules ofRobinia pseudoacacia growing in zinc-lead mine tailing. The phytoremediation assisted bythe two strains was also estimated in this study.The MICs of CCNWGS0286and CCNWGS0123to copper were2.8mM and2.5mM,while to zinc were3.0mM and2.0mM, respectively. Biosorptions of zinc by livingbiomasses of CCNWGS0286and CCNWGS0123were investigated under optimal conditionsat pH5.0, initial metal concentrations of100mg/L and a dose of1.0g/L. Kinetics modelssuggested there was more than one step involved in the Zn²⁺sorption process, while apseudo-second-order model was more suitable to describe the kinetic behavior accurately. Thefittings of Langmuir and Freundlich isotherms to experimental data for Zn²⁺sorption of thetwo strains were not exactly the same. However, they all belonged to the chemisorptionprocess. Moreover, more than70%Zn²⁺could be recovered from Zn²⁺-loaded biomasses atpH1.0. Carbonyl, amino, carboxyl and aromatic groups were responsible for the biosorptionof Zn²⁺by both CCNWGS0286and CCNWGS0123. In addition, cellular deformation,precipitate and damage might be involved in Zn²⁺resistance of the two strains.Plant growth promoting bacterium Agrobacterium tumefaciens CCNWGS0286（176）displayed both high metal resistance and enhanced the growth of Robinia plants in metalcontaminated environment. Genes putatively conferring zinc resistance were identified in thedraft genome of A. tumefaciens176. Among the7,600Tn5insertional mutants of A. tumefaciens176generated by transposon mutagenesis, nine zinc sensitive isolates werescreened individually, most of which showed great and specific sensitivity to Zn²⁺and Cd²⁺.In addition, interruption of a putative oligoketide cyclase/lipid transport protein in mutant15-6reduced IAA synthesis and also showed reduced Zn²⁺and Cd²⁺resistance. Analysis ofthe genome sequence and of transcription via RT-PCR combined with transposon genedisruptions revealed ZntA-4200and transcriptional regulator ZntR1played an important rolein zinc homeostasis of A. tumefaciens176. In greenhouse studies, R. pseudoacacia inoculatedwith A. tumefaciens176displayed a significant increase in biomass production compared toplant biomass without inoculation even in a zinc-contaminated environment. Moreover, IAAoverproduction by A. tumefaciens176with little affected by Zn²⁺was proved to be the mainreason to enhance plant growth under zinc contaminated environment. Interestingly, thedifferences in plant biomass improvement among A. tumefaciens176（ZnRIAA+）, A.tumefaciens C58（ZnRIAA-）, zinc sensitive mutants12-2（ZnSIAA+） and15-6（ZnSIAA-）revealed phytohormones were the dominant factor in enhancing plant growth in contaminatedsoil when compared to the influence of genes encoding zinc resistance determinants.Genes putatively conferring copper resistance were identified in the draft genome of M.amorphae CCNWGS0123（186）.10,000Tn5insertional mutants were generated bytransposon mutagenesis and eight copper sensitive isolates were obtained. Tn5insertion siteswere located on the whole genome of M. amorphae186, which were sorted into threecategories as P-type ATPase, hypothetical protein and others including GTP-binding proteinand ribosomal protein. Analysis of the genome sequence and of transcription via RT-PCRcombined with transposon gene disruptions revealed CopA-6910and CusB played animportant role in copper homeostasis of M. amorphae186. CopA-6910predominantly carriedout Cu+efflux from the cytoplasm to the periplasm, while CusAB was responsible for Cu+detoxification of the periplasm by export to extracellular space. Symbiotic nitrogen fixationwas proved to be the key point that helping plant growth ever under copper contaminatedenvironment. Moreover, M. amorphae186and its two mutants3-42（copA::Tn5） and34-28（ccmX::Tn5） were able to form symbiosis with R. pseudoacacia. However, nodule numbers,total N content, plant biomass and leghemoglobin expression were all reduced wheninoculated with mutant34-28（ccmX::Tn5）. Therefore, mutagenesis of hytothetical gene（ccmX） which is likely related with cbb3-Cox not only affected copper resistance, but alsoaffected symbiotic relationship with R. pseudoacacia. In greenhouse studies, R. pseudoacaciainoculated with M. amorphae186displayed a significant increase in biomass production, totalN content and copper accumulation compared to plant biomass without inoculation even in acopper-contaminated environment. Moreover, copper accumulation in roots was much higher than the accumulation in shoots, indicating the potential and safety when applyingrhizobia-legumes symbiosis for phytoremediation of metals.更多还原