【作者】 王艳；

【导师】 王小雨；

【作者基本信息】 东北师范大学 ， 生态学， 2017， 硕士

【摘要】 双组份信号系统是细菌中最常见的信号传导系统,能够感应、传导外界环境刺激,调节菌体内对应的基因适应性表达,在细菌抵抗不良环境的过程中发挥着重要的作用。Cupriavidus gilardii CR3是典型的重金属抗性基因位于染色体上的细菌,对铜的最小抑菌浓度（MIC）是3 mM。以往研究表明贪铜菌Cupriavidus metallidurans CH34、Cupriavidus necator N-1、Cupriavidus taiwanensis LMG 19424的双组份信号传导系统丰富多样、功能强大且参与重金属抗性调控机制。目前,关于C.gilardii CR3的双组份信号系统的研究尚无报道。本文首次研究了C.gilardii CR3全基因组的双组份信号系统分布特征,克隆鉴定了铜抗性双组份信号基因（copSR）,运用实时荧光定量PCR测定了不同铜离子浓度胁迫下copSR及其调控的结构基因（copB）在mRNA水平上的差异表达情况。此外,通过生物信息学技术对双组份蛋白CopSR进行了结构分析、功能预测和KEGG代谢通路分析。基于上述研究,本文分析了贪铜菌复杂多样的信号传导机制,揭示了C.gilardii CR3铜抗性调控机制。本文研究结果可为C.gilardii CR3在铜污染水体和土壤修复的潜在应用提供重要的理论参考。主要研究结果如下:（1）通过比较基因组学分析,结果表明C.gilardii CR3全基因组共有96个基因编码双组份信号蛋白,组成了22个双组份信号系统,其中与铜抗性相关的双组份信号系统比例最大。C.gilardii CR3的双组份信号系统与同属C.metallidurans CH34、C.necator N-1、C.taiwanensis LMG 19424拥有10个相同的HK蛋白功能域和7个相同的RR蛋白功能域,维持贪铜菌间的保守特性。此外,C.gilardii CR3含有4个HK特有蛋白功能域和1个RR特有蛋白功能域,特异性地适应C.gilardii CR3的生存环境。（2）克隆结果表明双组份信号系统编码基因copSR确实存在于C.gilardii CR3中。测序及NCBI序列比对结果表明,C.gilardii CR3中的copSR基因序列与标准copSR基因序列完全相同,copS为1404 bp,copR为687 bp。Smart数据库分析表明,copS编码的蛋白CopS为C.gilardii CR3的双组份感应蛋白CopS（WP₀53820945.1）,copR编码的蛋白CopR为C.gilardii CR3的双组份反应调节蛋白CopR（NP₂51499.1）。菌株CR3的CopS蛋白与Cupriavidus sp.HPC（L）的感应传导激酶同源性较高,菌株CR3的CopR蛋白与Cupriavidus sp.HPC（L）的调控因子同源性较高。（3）通过实时荧光定量PCR研究C.gilardii CR3在Cu2+刺激下目标基因copSRB的差异表达情况。结果表明,C.gilardii CR3在0.5 mM、1 mM、2 mM Cu2+胁迫2 h后,copSR表达量上调,且随着铜离子浓度增加而显著升高（P<0.001）,2 mM达到最大。C.gilardii CR3在不同浓度Cu2+胁迫下,各生长阶段的基因表达规律不尽相同。在0.5mM Cu2+胁迫下,copSR基因表达量在7 h（对数生长早期）达到最大;1 mM Cu2+胁迫时下copSR基因表达量在4 h（迟缓生长晚期）达到最大;2 mM Cu2+作用下copSR在2 h（迟缓生长早期）表达量达到最大。即铜离子浓度越高,copSR达到最大表达量的时间越短。copB的表达受copSR调控,与copSR表达趋势一致。（4）生物信息学分析表明,C.gilardii CR3的CopS为跨膜组氨酸激酶,具有功能域HATPase_c、HisKA和HAMP信号域,第255位氨基酸残基为保守的组氨酸残基,发生自磷酸化反应。C.gilardii CR3的Cop R为胞质调控蛋白,具有功能域REC和trans_reg_C,第51位为天冬氨酸残基,接收来自CopS的磷酸基团。C.gilardii CR3的CopS三级结构与Thermotoga maritima的反应调节蛋白DrrD结构相似,CopR蛋白三级结构与Caulobacter crescentus的组氨酸激酶DivL结构相似。综上,C.gilardii CR3含有丰富多样的双组份信号系统,C.gilardii CR3中的copSR为铜抗性双组份信号系统。铜抗性双组份蛋白CopS和CopR共同感应和传导C.gilardii CR3外界环境中铜离子刺激,调控下游铜抗性结构基因表达,维持菌体胞内铜离子平衡,对C.gilardii CR3抵抗外界环境中的铜离子威胁起到至关重要的作用。更多还原

【Abstract】 Two component signal system（TCS）is the most common signal transduction system in bacteria.TCS can sense the external environmental signals,and regulate the corresponding gene expression in the cell,which play an important role in the process of bacteria resistance to stress in the environment.Cupriavidus gilardii CR3 is a representative bacterium of heavy metal resistance gene on the chromosome.C.gilardii CR3 has high resistance to copper,of which minimum inhibitory concentration is 3 mM.The two-component signal transduction system of Cupriavidus metallidurans CH34,Cupriavidus necator N-1 and Cupriavidus taiwanensis LMG 19424 are various and involved in the regulation mechanism of heavy metal resistance.So far,there is no report on the two component signal system for Cupriavidus gilardii CR3.In this study,the distribution of two component signal system in C.gilardii CR3 genome was studied for the first time,and the predicted copSR was identified by cloning.Real-time fluorescence quantitative PCR was used to determine the expression of copSR and their regulated copB gene at mRNA level.In addition,the structure and function of protein CopSR were predicted by bioinformatics analysis,and the KEGG metabolic pathway was analyzed.Based on above research,we analyzed the complex and diverse signal transduction mechanism of Cupriavidus sp.,revealed copper resistance mechanism of C.gilardii CR3 and provided important theoretical reference for C.gilardii CR3’potential application in copper pollution remediation of water and soil.The main findings are as follows:（1）Through comparing the genomic analysis of the C.gilardii CR3 genome,we find that C.gilardii CR3 has 96 genes encoding two component signal proteins.C.gilardii CR3 has 22 two component signal systems,which copper resistance-related two component signal system has the largest proportion.The two component signaling system of strain CR3 was compared with C.metallidurans CH34,C.necator N-1 and C.taiwanensis LMG 19424.Because of the Cupriavidus sp’conservative nature,they share 10 high homologous HK protein functional domains and 7 high homologous RR protein domains.At the same time,C.gilardii CR3 contains four HK-specific protein functional domains and one RR-specific protein domain,specifically adapting to the unique habitat of C.gilardii CR3.（2）The clonal results indicated that the two component signal system copSR does exist in C.gilardii CR3.Sequencing and NCBI sequence comparison showed that the copSR gene sequence in C.gilardii CR3 was identical with the standard copSR gene sequence.copS was 1404 bp and copR was 687 bp.Smart database analysis showed that copS was sensing protein CopS（WP₀53820945.1）in C.gilardii CR3 and CopR was regulatory protein CopR（NP₂51499.1）in C.gilardii CR3.Protein CopS was homologous to sensing kinase in Cupriavidus sp.HPC（L）,copR was homologous to regulatory protein Cupriavidus sp.HPC（L）.（3）The expression of target gene in C.gilardii CR3 under Cu2+ stimulation was studied by real-time fluorescence quantitative PCR.The results showed that the expression of copSR was up-regulated under copper ion stress.Under 0.5 mM,1 mM and 2 mM Cu2+ stress for 2 h,the expression of copSR increased with the increasion of copper ion concentration.The expression of copSR reached the maximum at 2 mM.Different gene expression pattern of C.gilardii CR3 was found under different copper concentrations during growth stage.The copSR reached the maximum expression at 7 h under 0.5 mM Cu2+stress.Gene copSR reached the maximum expression at 4 h under 1 mM Cu2+stress.The expression of copSR reached maximum at 2 h under Cu2+stress.Obviously,the higher concentration of copper,the time for copSR to achieve the maximum expression was shorter.Gene copB expression is consistent with the expression of copSR,which was regulated by copSR.（4）Bioinformatics analysis showed that CopS is transmembrane histidine kinase with functional domain HATPase_c,HisKA,and HAMP signal domains.The 255 th amino acid residue is a conserved histidine residue and undergoes autophosphorylation.CopR is cytoplasmic regulatory protein with functional domain REC and trans_reg_C,and at position 51 is an aspartic acid residue,where received phosphate group from CopS.CopS tertiary structure is similar to the DrrD structure in Thermotoga maritima.The structure of the CopR protein is similar to structure of Caulobacter crescentus DivL.In summary,C.gilardii CR3 contains a rich and diverse two component signaling system.The copSR in C.gilardii CR3 is a copper-resistant two component signaling system.Copper-resistant two-component protein CopS and CopR co-induction and conduction external environment of copper ion stimulation in C.gilardii CR3.Then CopR regulate the expression of downstream copper resistance structure gene copB,and maintain the intracellular copper ion balance,which allow C.gilardii CR3 resist high concentrations of copper ion stress.更多还原