基于苋菜转录组的ARF基因家族鉴定及表达

ARFs(auxin response factors)是重要的生长素响应因子,在植物的生长发育过程中起着关键的作用.基于'大红'苋菜转录组数据,通过在线分析软件SMART和NCBI-Blastp的注释筛选苋菜ARF基因家族成员(AtrARF);利用生物信息学分析软件,对AtrARFs蛋白的理化性质、二级结构、亚细胞定...     

Simple and rapid determination of dioxin in fish and sea food using a highly sensitive reporter cell line, CBG 2.8D

Food, especially animal origin food is the main source of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), and dioxin-like polychlorinated biphenyls (dl-PCBs) for human exposure. So, a simple, rapid and cheap bioassay method is needed for determination of dioxins in food samples. In this study, we used a new highly sensitive reporter cell line to determine the concentration of dioxins in 33 fish and seafood samples. The samples were extracted by shaking with water/isopropanol (1:1 v/v) and hexane and cleaned-up by a multi layered silica gel column and an alumina column, then analyzed using CBG 2.8D cell line. We compared the results obtained from the CBG 2.8D cell assay to those obtained from conventional High-Resolution Gas Chromatography–High Resolution Mass Spectrometry (HRGC–HRMS) analysis. Good correlations were observed between these two methods (r²=0.93). While the slope of regression line was 1.76, the bioanalytical equivalent (BEQ) values were 1.76 folds higher than WHO-TEQ values and the conversion coefficient was 0.568 (the reciprocal of 1.76). In conclusion, CBG 2.8D cell assay was an applicable method to determine dioxins levels in fish and sea food samples.     

全氟辛烷磺化物(PFOS)诱导斑马鱼胚胎p53基因的点突变

将斑马鱼胚胎于0μg·1-1(空白对照),5μg·l-1,16μg·l-1和50μg·l-1全氟辛烷磺化物(PFOS)溶液中暴露染毒5h,采用变性高效液相色谱方法对PCR扩增的含斑马鱼胚胎p53基因外显子7的173 bp的目标片段进行突变分析.结果显示,16μg·l-1和50μg·l-1两个处理组与对照组相比均有显著性差异,表明PFOS可以诱导斑马鱼胚胎p53基因外显子7的点突变.     

重组BALB/C鼠冷诱导RNA结合蛋白CIRP多克隆抗体的制备与鉴定

冷诱导RNA结合蛋白(CIRP)是第一种在哺乳动物细胞中被发现的冷休克蛋白,伴随着细胞冷应激过程过量表达.为进一步研究冷诱导RNA结合蛋白CIRP的生物学功能,构建重组BALB/C鼠CIRP的原核表达载体,重组BALB/C鼠CIRP蛋白经NI-NTA亲和树脂纯化后免疫獭兔制备抗CIRP多克隆抗体,以间接ELISA和Western blot检测抗体效价和抗原特异性.本实验成功构建了CIRP的原核表达载体,在E coli XL-1-blue得到了高效表达,利用原核表达包涵体蛋白免疫獭兔获得了高效价的CIRP多克隆抗体,该抗体能识别肝脏和睾丸中天然的CIRP蛋白.图5参14     

近江牡蛎4种类型GST基因cDNA全序列的克隆与分析

为从分子水平上研究不同类型谷胱甘肽S-转移酶(Glutathione S-transferase,GST)基因在近江牡蛎(Crassostrea ariakensis)等贝类体内代谢去毒过程中的作用,采用RT-PCR及RACE法,分离、克隆得到近江牡蛎肝脏4种类型GST基因cDNA全序列.序列分析结果表明,mu、pi、omega、sigma4种类型GST基因cDNA全长分别为970bp、773bp、999bp、1277bp,分别编码215、207、242、203个氨基酸.构建系统进化树发现,除太平洋牡蛎(Crassostrea gigas)pi型GST外,所有类型GSTs序列都能各自聚集成一簇.     

嗜热脂肪土芽孢杆菌木聚糖酶基因的合成及其在大肠杆菌中的表达

来自嗜热脂肪土芽孢杆菌的木聚糖内切酶XT6在工业上有着重要的应用,已经成功应用于工业规模的生产试验.本文作者在合成XT6基因全序列的同时对其密码子进行了优化,且构建重组质粒在大肠杆菌中高表达.通过优化表达条件,功能正常的XT6基因在大肠杆菌中成功过量表达,蛋白表达量占细胞中总蛋白的65%.重组表达的木聚糖内切酶XT6特性和天然酶相似,以桦木木聚糖为底物测定细胞提取物中木聚糖酶活性,最大活性高达3 030 U/mL.本文首次报道了来自嗜热脂肪土芽孢杆菌中木聚糖酶基因全序列的合成和在大肠杆菌中成功过量表达.图6表1参19     

人体肠道耐药基因组的研究进展

人类肠道菌群是耐药基因(antibiotic resistance genes,ARGs)的"储存库",且与人体健康密切相关。目前,抗生素的滥用严重,进一步加剧了耐药基因的传播和扩散。细菌耐药问题严重影响人体健康、食品安全和生态安全,携带耐药基因的致病菌对临床治疗造成巨大威胁。本文结合国内外研究进展,在总结人体肠道耐药基因组研究方法的基础上,探讨了肠道耐药基因的组成、来源、传播和进化,并对未来研究进行了展望,旨在促进公众对肠道耐药基因的认知,并为抗生素的合理使用提供理论支持。     

抗生素单一及联合暴露对RP4质粒介导的抗性基因水平转移的hormesis效应研究

抗生素滥用导致的抗性基因(ARGs)泛滥问题引起人们的日益关注。目前关于抗性基因的研究主要集中在环境监测方面,但是关于抗生素对ARGs传播的影响研究还相对缺乏。质粒是ARGs传播的重要载体,因此以基于RP4质粒的大肠杆菌(E.coli)接合转移体系为研究对象,探讨了盐酸四环素、甲氧苄啶、磺胺氯哒嗪、磺胺异唑在单一暴露条件下对RP4质粒接合转移的影响。根据单一暴露的结果设计混合抗生素的浓度比,探究抗生素联合暴露实验对质粒接合转移的影响。结果表明:在单一暴露条件下,只有盐酸四环素对含有四环素抗性的RP4质粒的接合转移频率有低促高抑的hormesis效应;在联合暴露条件下,对RP4质粒接合转移无促进作用的抗生素会抑制四环素的hormesis效应,这一定程度上降低了抗生素的环境风险。并且用受体理论解释了抗生素对质粒接合转移的hormesis效应产生机制,为目前尚无定论的hormesis受体理论机制提供了证据支持。     

Recombinant human AhR-mediated GUS reporter gene assays for PCB congeners in transgenic tobacco plants in comparison with recombinant mouse and guinea pig AhRs

Four expression plasmids for recombinant human aryl hydrocarbon receptor (hAhR) consisting of a ligand binding domain of hAhR, a DNA-binding domain of LexA and a transactivation domain of VP16 as well as β-glucuronidase (GUS) reporter genes were constructed. All the expression plasmids were transformed into tobacco plants. The selected transgenic tobacco plants were used to assay. PCB congeners showed GUS activity in a TEF-dependent manner. The selected transgenic tobacco plant XhD4V17 was compared with the transgenic tobacco plants XmD4V26 and XgD2V23 containing recombinant mouse (m) AhR-mediated GUS reporter gene expression cassette and recombinant guinea pig (g) AhR-mediated GUS reporter gene expression cassette for PCB congener-inducible GUS activity. The data revealed that the tobacco plant XgD2V23 was the most active in PCB congener-inducible GUS activity. In a 1:1 mixture of PCB126 and PCB80 a reduced PCB126-induced GUS activity was observed in plant XgD2V23, which could possibly be due to interaction between PCB126 and PCB80.