大肠杆菌NrfA蛋白表达、纯化及多克隆抗体的制备

[目的]克隆大肠杆菌NrfA基因,构建pET-28a(+)-NrfA表达载体,制备相应的多克隆抗体并对其进行鉴定。[方法]以大肠杆菌基因组DNA为模板,PCR扩增得到NrfA基因编码区,构建pET-28a(+)-NrfA表达载体;经IPTG诱导表达并纯化重组蛋白;再免疫新西兰雄兔,制备多克隆抗体;用ELISA方法检测抗体的效价,Western Blotting检测抗体的特异性。[结果]构建的表达载体pET-28a(+)-NrfA在大肠杆菌中诱导后可高效表达NrfA蛋白;免疫获得的多克隆抗体用ELISA检测,其效价为1∶204 900;经Western Blotting分析,抗体的特异性较好。[结论]成功克隆大肠杆菌的NrfA基因,并构建了其表达载体,制备的NrfA多克隆抗体具有较高的效价和良好的特异性,为研究细菌有关NrfA奠定了基础。

Expression and Purification of E.coli NrfA Protein and Preparation of Polyclonal Antibody against NrfA

[Objective] This study aimed to clone the E. coil NrfA gene and construct the pET-28a （＋）-NrfA prokaryotic expression vector for preparation of polyclonal anti- body against E. coil NrfA. [Method] E. coil NrfA gene was cloned from the E. coli genome DNA by PCR and inserted into the vector pET-28a（＋） to construct prokary- otic expression vector pET-28a （＋）-NrfA. E. coil NrfA protein was expressed by IPTG induction and purified. Polyclonal antibody against NrfA protein was prepared by im- munizing rabbit with routine method. The specificity and titer of polyclonal antibody was confirmed by ELISA and Western Blotting. [Result] The constructed prokaryotic expression vector pET-28a（＋）-NrfA was induced by IPTG, the recombinant NrfA pro- tein could be expressed effectively. The titer of rabbit anti-NrfA polyclonal antibody obtained by immunization and purification was about 1：204 900. Western Blotting anal- ysis indicated that the obtained polyclonal antibody against E. coil NrfA protein had high titer and high specificity. [Conclusion] E. coil NrfA gene was cloned and the prokaryotic expression vector pET-28a （＋）-NrfA was constructed successfully, poly- clonal antibody with high titer and high specificity was prepared, which laid the foun- dation for the study of NrfA in different strains of bacteria.

稻田硝酸盐异化还原成铵细菌群落结构的垂向分布特性

硝酸盐异化还原成铵(Dissimilatory nitrate reduction to ammonium,DNRA)过程,可将土壤中的NO-3/NO-2还原成NH+4-N以被作物吸收利用,有助于土壤中氮素的保存.选择位于广东韶关市和吉林安图县的两块稻田为研究对象,通过分子生物学方法和高通量测序技术,深入探究了两种稻田土壤(0~1 m)中DNRA细菌丰度和群落结构的垂向分布特性.结果显示:两种稻田土壤中DNRA细菌更多的存在于表层土壤(0~20 cm)中,丰度最低值均出现在深层土壤(90~100 cm)中,且表层样品群落结构的多样性大于深层样品;两个稻田样品间的群落结构体现出明显的空间异质性,安图稻田样品DNRA细菌的丰度和群落结构多样性均大于韶关稻田样品;其中,相对丰度较高的Anaeromyxobacter(28.67%)、Caldimicrobium(19.49%)、Nitrospira(10.90%)和Chthoniobacter(9.15%)是两个稻田中DNRA细菌群落组成中的关键菌属.相关性分析表明,总硫(TS)、有机质(TOM)、含水率(MC)、碳氮比(C/N)与DNRA细菌丰度之间均有显著正相关性(p<0.01,n=20),偏碱性、有机质丰富、氮源缺乏、碳源丰富且C/N较高的环境及适宜的含水率是稻田垂向生态系统中DNRA细菌适宜的生存环境.

旱地作物根际和非根际土壤硝酸盐异化还原成铵细菌群落组成的研究

为研究典型旱地农田土壤硝酸盐异化还原成铵过程(Dissimilatory nitrate reduction to ammonium,DNRA)的群落组成,针对DNRA过程的功能基因nrfA进行高通量测序.根际和非根际、4种典型农作物共16个样品,质控后每个样品得到87000条序列,在相似度≥90%下划分到27952个OTUs,选取其中丰度较高的258个代表OTUs进行生态学分析.多样性分析(OTUs水平)结果表明:3/4的作物根际土壤样品中的DNRA群落丰富度、物种多样性和物种均匀度高于相应非根际样品,对比4种作物,粟作物根部土壤DNRA群落多样性最高,玉米作物非根际土壤最低.对代表OTUs进行分类,共定义到6个门(Phylum),19个属(Genus).其中相对丰度最高的3个属为Hyalangium(29.31%)、Chthoniobacter(20.33%)和Nitrospira(13.41%),表明三者在群落组成中占主导地位.结合土壤理化因子分析,DNRA群落相对丰度与NO^-_2-N、TN、含水率、TOM、pH及温度呈显著相关关系.本研究在一定程度上揭示了旱地农田土壤DNRA细菌的群落组成、多样性及与土壤环境因子的关系,为提高氮肥的利用效率和减小环境污染提供理论依据.

典型湿地沉积物中硝酸盐异化还原成铵的细菌群落结构的研究

为研究中国典型湿地沉积物硝酸盐异化还原成铵过程(Dissimilatory nitrate reduction to ammonium,DNRA)的群落组成,针对DNRA过程的功能基因nrfA进行高通量测序.选取中国典型湿地岸边带的表层沉积物8个样点,质控后每个样品得到60000条序列,在相似度≥90%得到279个OTUs进行生态学分析.由基因丰度值显示:8个湿地沉积物的丰度为(6.69±0.28)×10^7^(8.44±0.48)×10^8 copies g^-1.多样性分析(OTUs水平)结果表明:本研究的湿地沉积物样点中,南方湿地沉积物样点的多样性要高于北方样点.对代表OTUs进行分类,共定义到8个门(Phylum),23个属(Genus).其中相对丰度最高的3个属为Anaeromyxobacter(24.71%)、Anaerolinea(9.70%)和Dokdonella(7.94%),表明三者在群落组成中占主导地位.PCoA分析(OTUs水平)表明南北方地区差异是导致中国湿地沉积物中DNRA菌群结构不同的最主要影响因素.结合沉积物理化因子分析,DNRA细菌的丰度与碳氮比、年平均降水量及年平均温度呈显著正相关.本研究在一定程度上揭示了中国典型湿地沉积物DNRA细菌的群落组成、多样性及其与环境因子的关系.