【作者】 李哲斐；

【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】 根瘤菌是能与豆科植物共生固氮的重要农业微生物，若长期生长于重金属污染的土壤中，根瘤菌细胞会对重金属离子产生相应的抗性。本实验室从陕西凤县金属尾矿生长的天蓝苜蓿中分离到的S. meliloti CCNWSX0020在TY培养基中能够抵抗1.4mM的Cu²⁺。通过在含有0.5mM的Cu²⁺和不含Cu²⁺的TY液体培养中培养菌体，利用cDNA-AFLP技术分析了两种培养条件下的差异基因表达，最终获得了3条DNA差异片段（转录衍生片段TDF）。通过比对发现TDF1所在基因编码的蛋白与S. meliloti1021的铜离子外排P-typeATPase相似性达到97%；TDF2所在基因编码的蛋白与CandidatusAccumulibacter phosphatis clade IIA str. UW-1的RNase H同源性达到42%；TDF3是omp基因的一部分，omp基因含1446个碱基，编码含481个氨基酸的假定蛋白质，它和Rhizobium leguminosarum bv. viciae3841的外膜蛋白有75%的相似性。根据omp基因序列设计引物并从S. meliloti CCNWSX0020基因组中扩增出omp基因全长，以插入失活的方式构建omp基因敲除载体，通过同源重组得到突变体。0.8mM的Cu²⁺能明显抑制该突变体的生长，同时突变对Cu²⁺、Zn²⁺、Pb²⁺等的抗性下降，证实了omp基因与S. meliloti CCNWSX0020的铜抗性有关。PCR扩增得到含启动子的omp基因全长，将其克隆到广宿主质粒pBBR1MCS-5上，以三亲杂交将其导入到突变株中，结果发现omp基因能使突变体的抗铜能力恢复到野生型的80%左右，进一步证明omp基因与铜抗性相关。在omp基因上游有两个基因SM0020₁8782和SM0020₁8787编码未知功能的假定蛋白，在omp基因的下游有另外三个基因SM0020₁8797、M0020₁8802和SM0020₁8807分别编码多铜氧化酶、假定的铜离子结合蛋白和一个假定的铜伴侣分子，这6个基因构成了操纵子，它们对细胞的铜抗性起到了重要作用。以添加0.5mM铜离子和不含铜的TY培养基培养CCNWSX0020野生型和omp突变体菌株，利用GC-MS测定从细胞中提取出的代谢产物。通过PCA、PLS、TICL等对数据进行分析，结果显示，在两种培养条件下，细胞内产生的代谢物有明显的差异。在铜离子的诱导下，突变体中丙氨酸、甘氨酸、亮氨酸和缬氨酸等浓度增加，这样加速蛋白质的合成并有利于修复突变体中由于铜离子积累所引发的蛋白损伤。在野生型细胞中苹果酸、果糖、甘油-3-磷酸等含量增加，它们通过调整TCA循环来积累抗氧化物：还原性辅酶Ⅱ（NADPH）。同时，两种菌体在含铜离子培养基中都可以产生有助于细胞抵抗铜离子毒性的吲哚-3-乙酸（IAA）、超氧化物歧化酶（SOD）及过氧化氢酶（CAT）。但是同野生型相比，突变体中SOD及CAT在较低的铜离子浓度下活性达到最大，说明突变体对铜离子较敏感，因此铜离子引发的自由基可能造成某些酶活性改变，因而突变体所产生的IAA要低于野生型细胞。通过全基因测序发现S. meliloti CCNWSX0020基因组中共包含7,001,588个碱基，序列中G+C的含量为59.9%，通过拼接，将碱基数大于500bp的序列予以保留，共得到了233个contig。基因组中一共包含有7086个基因，其中有6个基因编码rRNA，47个基因编码tRNA，7033个基因编码蛋白质。对有功能注释的基因进行分析发现，在S.meliloti CCNWSX0020中包含有与细胞重金属抗性有关的基因51个；在基因组中包含有多个基因与抗氧化物SOD、CAT、谷胱甘肽（GSH）的代谢有关；另外还有一些基因编码的蛋白与ACC脱氨酶、海藻糖、IAA等的合成有关，CCNWSX0020通过产生这类物质促进植物生长并增加其生物量以吸收更多铜离子，从而使土壤中铜离子浓度降低，增强菌体对铜离子的抗性。MerR-like突变体中，通过荧光定量对6个编码P-typeATPase的基因表达进行检测，结果表明SM0020₀5727和SM0020₀5862两个基因受MerR-like转录因子的调控。通过序列比对发现这两个基因编码的P-type ATPase N末端没有金属结合结构域，第6跨膜区没有保守的CPC蛋白模体，但它含有TCP序列，这与已经报道的P-typeATPase明显不同，因此它有可能是一种比较特殊的P-typeATPase。更多还原

【Abstract】 Rhizobia are important agricultural microorganisms which can fix nitrogen by symbiosewith legumes. If long-term growth in the heavy metal polluted soil, Rhizobium cells may beform some mechanism resistant to heavy metal ions. Sinorhizobium. meliloti CCNWSX0020,isolated from root nodules of Medicago lupulina growing in Fengxian gold mine tailings inthe northwest of China, was resistant up to1.4mM Cu²⁺in tryptone-yeast （TY） extractmedium. By culturing the bacteria in the containing0.5mM Cu²⁺and Cu²⁺-free TY medium,the difference gene expression were analyzed by using cDNA-AFLP technique and obtained3differential transcript Derived Fragments. The differential fragments were sequenced andanalyzed with BLAST, the result showed that the TDF1corresponding gene encodes a proteinwith97%similarity to copper ion efflux P-type ATPase of S. meliloti1021; The TDF2corresponding gene encodes a protein with42%identity to RNase H of CandidatusAccumulibacter phosphatis clade IIA str. UW-1; The TDF3is a part of omp gene, omp contains1446nucleotides which encodes a putative protein with75%similarity to outer membraneprotein of Rhizobium leguminosarum bv. viciae3841.omp gene was amplified from S. meliloti CCNWSX0020and built omp gene knockoutvector.0.8mM Cu²⁺ could significantly inhibit the growth of the homologous recombinationmutant. The omp gene was cloned into pBBR1MCS-5and transferred into mutant. The resultshowed that omp gene can restore about80%copper resistance. Two genes SM0020₁8782and SM0020₁8787encode two unknown functions putative proteins in the upstream of Ompgene, there are three other genes SM0020₁8797, SM0020₁8802and SM0020₁8807encodecopper ion multicopper oxidase, the putative binding protein and a putative copper chaperonein the downstream of the omp gene, these6genes constitute an operon which plays animportant role in copper resistance.The metabolites of CCNWSX0020and omp mutant growing in TY medium with andwithout0.5mM copper ions were determined by GC-MS. The data were analyzed using PCA,PLS and TICL, the results showed that intracellular metabolites of cells cultured in mediumwith and without copper have obvious difference. The metabolites of wild type and mutanthave certain difference induced by copper, the concentration of alanine, glycine, leucine andvaline increased in mutant which can accelerate protein synthesis and repair damage proteincaused by copper. Malic acid, fructose, glycerol-3-phosphate content increased in wild-typecells which can accumulate antioxidant NADPH by adjusting the TCA. At the same time, two kinds of bacteria can produce IAA, SOD and CAT in the medium with copper. But comparedwith wild-type, the CAT, SOD activity of mutant reaches the maximum under lowconcentration copper, it showed mutant was more sensitive to copper, so copper lead freeradicals may cause inactivation of some enzyme and decreased the IAA yield in mutant.The draft genome sequence of Sinorhizobium meliloti CCNWSX0020comprises7,001,588bases. The genome of S. meliloti CCNWSX0020has a G+C content of59.9%.There are a total of7,086genes, including6rRNA genes,47tRNA genes, and7,033putativeprotein-coding sequences. Additionally, Sinorhizobium meliloti CCNWSX0020carried51predicted protein-coding genes involved in heavy metal resistance. Multiple genes relate toantioxidant SOD, CAT, GSH metabolism have been found in CCNWSX0020genome; Thereare some other genes encode proteins relate to synthesis of ACC deaminase, trehalose, IAA,this kind of metabolites produced by CCNWSX0020can promote plant growth and increasethe biomass to absorb more copper ions, thus reduce the copper concentration in the soil andenhance tolerance of bacteria to copper. The expression of six genes encoding P-type ATPasewere detected by RT-PCR in the MerR-like mutant, the results show that the SM0020₀5727and SM0020₀5862genes were regulated by MerR-like transcription factor. However, theP-type ATPases encode by these genes are not contain N-terminal CXXC motif. In addition,the gene product of SM0020₀5862probably is a special P-type ATPase since it contains nota CPC motif but TCP which is highly unusual.更多还原