海岸带生物学与生物资源保护实验室知识库

滨海滩涂是海岸带潮间浅滩，是丰富的土地资源，而滨海盐渍化越来越严重，研究耐盐植物-微生物互作及联合修复具有理论和实际意义。茎瘤固氮根瘤菌（Azorhizobium caulinodans）ORS571 属于α-变形菌，能够在宿主滨海耐盐植物毛萼田菁（Sesbania rostrata）根部和茎上共生结瘤，也可自生或作为内生菌在其他植物体内固氮。毛萼田菁具有强的耐盐、耐涝能力，常作为滩涂盐碱地土壤改造的先锋植物，在滩涂资源改良利用方面有巨大应用潜力。毛萼田菁也具有较高的经济价值，可用于制作饲料和食品稳定剂等。趋化作用是细菌感受外界化学浓度梯度，向利于自身生长的环境运动的应答过程。在细菌的趋化信号通路中，趋化受体位于起始部分，分为跨膜趋化受体和可溶性趋化受体，跨膜趋化受体主要负责感受胞外信号。本文选取三个茎瘤固氮根瘤菌ORS571 跨膜趋化受体TlpA1、AZC_0719、AZC_1721，对其感应机理进行初步探究，有助于从分子层面阐释茎瘤固氮根瘤菌ORS571 与其宿主毛萼田菁的共生机制。主要的研究内容与结果如下：
利用生物信息学的手段对茎瘤固氮根瘤菌ORS571 趋化受体蛋白质结构域进行分析，发现ORS571 共有43 个趋化受体，其中37 个为跨膜趋化受体。TlpA1位于趋化基因簇前端，具有明显的跨膜区，但其胞外感应结构域尚未知。AZC_0719 具有4HB_MCP 结构域，该结构域普遍存在于原核生物信号转导蛋白中，其两端多伴随跨膜结构域，只感应胞外信号，趋化物类型主要为有机酸类和氨基酸类。AZC_1721 具有CHASE3 结构域，该结构域是位于胞外的感应结构
域，存在于组氨酸激酶、趋化受体中等，主要参与细菌趋化及代谢途径。蛋白质结合位点预测显示TlpA1、AZC_0719、AZC_1721 N 端胞外部分均具有完整的蛋白质结合位点，且三个结合位点预测都与大肠杆菌K12 的组氨酸激酶NarQ 胞外部分同源性最高，NarQ 感应硝酸盐、亚硝酸盐、脂类及碳类等。
本文构建了跨膜趋化受体蛋白TlpA1、AZC_0719、AZC_1721 的N 端表达株，PCR 与基因测序结果显示，表达株构建成功。诱导表达目的蛋白，SDS-PAGE电泳结果显示，表达的目的蛋白为可溶性蛋白。等温滴定量热法（ITC）与蛋白
质热稳定性试验结果显示，TlpA1 与琥珀酸之间存在相互作用，AZC_1721 与3-羟基-2 丁酮之间存在相互作用。
突变株ΔTlpA1 由本实验室提供。本文构建了缺失突变株ΔAZC_0719 和ΔAZC_1721，并对三株突变株进行生长速率、趋化性行为、胞外多糖、生物膜形成、絮凝等表型检测。结果显示，三株突变株与野生型在生长速率方面无显著性
差异。趋化性行为检测结果显示，在碳源为3-羟基-2-丁酮的半固体趋化板上，突变株ΔAZC_1721 的趋化圈显著性大于野生型。在不同碳源的半固体趋化板上，突变株ΔAZC_0719 的趋化圈与野生型无显著性差异。胞外多糖试验结果显示，突变株ΔAZC_0719 和ΔAZC_1721 在刚果红平板上的菌落形态及胞外多糖的分泌与野生型相比无显著性差异。生物膜形成试验结果显示，在外界存在氮源或固氮条件下，三株突变株ΔTlpA1、ΔAZC_0719 和ΔAZC_1721 生物膜形成能力低于野生型。絮凝结果显示，三株突变株ΔTlpA1、ΔAZC_0719、ΔAZC_1721 与野生型无显著性差异。

1.8 本文选题背景及主要研究内容 ................................................................16
1.8.1 选题背景 ..................................................................................................16
1.8.2 选题目的及意义 ......................................................................................16
1.8.3 主要研究内容 ..........................................................................................17
1.8.4 技术路线 ..................................................................................................17
第2 章 茎瘤固氮根瘤菌跨膜趋化受体蛋白TlpA1 功能分析 .....................19
2.1 试验材料 ......................................................................................................19
2.1.1 试验菌株与质粒 ......................................................................................19
2.1.2 试剂与仪器 ..............................................................................................19
2.1.3 培养条件 ..................................................................................................19
2.2 试验方法 ......................................................................................................20
2.2.1 生物信息学分析 ......................................................................................20
2.2.2 茎瘤固氮根瘤菌ORS571 总DNA 提取 ..............................................20
2.2.3 引物设计 ..................................................................................................21
2.2.4 N 端表达载体的构建 ..............................................................................21
2.2.5 N 端表达菌株的构建 ..............................................................................25
2.2.6 原核表达 ...................................................................................................25
2.2.7 蛋白分离与纯化 .......................................................................................26
2.2.8 等温滴定量热法（ITC）试验 ................................................................27
2.2.9 生物膜实验 ...............................................................................................27
2.2.10 絮凝实验 .................................................................................................27
2.3 结果与分析 ...................................................................................................28
2.3.1 TlpA1 蛋白结构域的预测 .....................................................................28
2.3.2 TlpA1 N 端表达菌株的构建 ..................................................................29
2.3.3 原核表达结果 ..........................................................................................29
2.3.4 等温滴定量热法(ITC)试验结果 .............................................................30
2.3.5 生物膜试验结果 ......................................................................................31
2.3.6 絮凝试验结果 ..........................................................................................32
2.4 讨论 ...............................................................................................................33
第3 章 茎瘤固氮根瘤菌跨膜趋化受体蛋白AZC_0719 和AZC_1721
的功能分析 ..........................................................................................................34
3.1 试验材料 .......................................................................................................34
3.1.1 菌株与质粒 ................................................................................................34
3.1.2 试剂与主要仪器 .......................................................................................34
3.2 试验方法 .......................................................................................................35
3.2.1 引物设计 ...................................................................................................35
3.2.2 N 端表达载体的构建 ..............................................................................35
3.2.3 蛋白质热稳定性试验 ..............................................................................35
3.2.4 缺失突变载体构建 ..................................................................................36
3.2.5 缺失突变株ΔAZC_0719/ΔAZC_1721 的构建 .................................36
3.2.6 生长速率的测定 ......................................................................................38
3.2.7 趋化实验 ..................................................................................................38
3.2.8 胞外多糖、生物膜及絮凝能力的观察与测定 ....................................38
3.3 结果与分析 ...................................................................................................38
3.3.1 AZC_0719/AZC_1721 蛋白质结构域分析与结合位点预测 ...........38
3.3.2 AZC_0719、AZC_1721N 端表达菌株的构建 ..................................40
3.3.3 原核表达 ...................................................................................................41
3.3.4 蛋白质热稳定性试验 ..............................................................................41
3.3.5 AZC_0719、AZC_1721 缺失突变株的构建 .....................................43
3.3.6 生长速率测定 ..........................................................................................44
3.3.7 趋化性行为检测结果 ..............................................................................44
3.3.8 胞外多糖的观察结果 ..............................................................................44
3.3.9 生物膜形成与定量分析 ..........................................................................45
3.3.10 絮凝试验结果 ........................................................................................46
3.4 讨论 ..............................................................................................................46
第4 章 全文总结及展望 ...................................................................................49
4.1 全文总结 ......................................................................................................49
4.2 创新点 ..........................................................................................................50
4.3 展望 ..............................................................................................................50
参考文献 .............................................................................................................53
附 录 ....................................................................................................................61

Tidal flat is the intertidal shoal in the coastal zone, which is rich in land resources.Coastal salinization is becoming more and more serious, and it is the oretical and practical significance to study salt-tolerant plant-microorganism interaction and restoration. Azorhizobium caulinodans ORS571 belongs to alpha-subdivision of proteobacteria that symbiotic nodulated on the roots and stems of its host coastal salt tolerant plant Sesbania rostrata, and can also fix nitrogen during free-living growth or as an endophyte in other plants. S. rostrata has strong resistance to salt and waterlogging. It is used as a pioneer plant in the phytoremediation of saline and alkaline soil in tidal flat and has great potential in the improvement and utilization of tidal flat resources. S. rostrata also has high economic value and could be used in making feed and food stabilizer. Chemotaxis is a response process in which bacteria sense the chemical gradients and move toward an environment conducive to their growth. In the chemotactic signaling pathway of bacteria, chemoreceptors are located in the initial part, which contain transmembrane chemoreceptors and soluble chemoreceptors. Transmembrane chemoreceptors are mainly responsible for sensing extracellular signals. Three transmembrane chemoreceptors TlpA1, AZC_0719 and AZC_1721 of A. caulinodans ORS571 are selected in this study to explore the sensing mechanism, which is helpful to explain the symbiosis mechanism of A. caulinodans ORS571 and its host S. rostrata from the molecular level.The main results were as follows:

Domain analysis showed that there were 43 chemoreceptors in A. caulinodans ORS571, 37 of which were transmembrane chemoreceptors. TlpA1 was located at the front of the chemotaxis gene cluster and had obvious transmembrane region, but its extracellular sensing domain was unknown. AZC_0719 had a 4HB_MCP domain. It is generally found in prokaryotic signal transduction proteins. Both ends of the domain are accompanied by transmembrane domains, and only extracellular signals are sensed. The chemoattractants are mainly organic acids and amino acids. AZC_1721 had a CHASE3 domain, which was an extracellular sensing domain that existed in histone kinase, MCPs, etc. It was involved in bacterial chemotaxis and metabolic pathways. Protein binding site prediction showed that TlpA1, AZC_0719 and AZC_1721 Nterminal
extracellular parts all had complete protein binding sites, and the three binding sites predicted had the highest homology with the extracellular part of E. coli K12 histidine kinase NarQ, which is sensing nitrate or nitrite, lipid and carbon.
The N-terminal expression strains of TlpA1, AZC_0719 and AZC_1721 were constructed. PCR and gene sequencing results showed that the expression strain was successfully constructed. SDS-PAGE electrophoresis results showed that the target protein was soluble. Isothermal titration calorimetry (ITC) and protein thermal shift assay results showed that there was interaction between TlpA1 and succinic acid, and between AZC_1721 and 3-hydroxy-2-butanone.
Mutant strain ΔTlpA1 was provided by our laboratory. The mutant strains ΔAZC_0719 and ΔAZC_1721 were constructed by this study. For mutants ΔTlpA1, ΔAZC_0719 and ΔAZC_1721, the growth rate, chemotaxis behavior, extracellular polysaccharide, biofilm formation, flocculation rate were detected. The results showed that there was no significant difference in the growth rate between the mutant and the wild type. Chemotaxis behavior test results showed the mutant strains ΔAZC_1721 fomed the chemotaxis ring significantly greater than the wild type, when 3-hydroxy-2-butanone used as carbon source. But at semisolid plates with different carbon source, the mutant strain ΔAZC_0719 formed the same chemotaxis ring with the wild type. The exopolysaccharide test results showed that mutant strains ΔAZC_0719 and ΔAZC_1721 had no significant difference in Congo red colony morphology and the secretion of extracellular polysaccharide compared with wild type. Biofilm formation test results showed that the biofilm formation ability of three mutant strains ΔTlpA1, ΔAZC_0719 and ΔAZC_1721 is significantly lower than the wild type in the presence or absence nitrogen source. Flocculation test results showed there was no significant difference between three mutant strains ΔTlpA1, ΔAZC_0719, ΔAZC_1721 with wild type. Keywords: A.caulinodans ORS571, transmembrane chemoreceptor, chemotaxin, Chemotaxis behavior