亚硝态氮胁迫下菲律宾蛤仔实时定量P CR内参基因的筛选

为了更好地从基因角度研究亚硝态氮对贝类的生态毒性,需要筛选一个合适的内参基因作为参考,对其毒性相关基因进行定量表达分析。本研究以菲律宾蛤仔(Ruditapes philippinarum)作为研究对象,以亚硝态氮胁迫的鳃组织cDNA作为模板,利用geNorm软件和NormFinder软件对6个候选内参基因进行表达稳定性分析,并对2个结果做了综合赋权分析。结果显示,geNorm分析的基因表达稳定性结果为ActinCyPAEF1αUbi18STubu,NormFinder软件分析的结果为ActinUbiCyPAEF1α18STubu,最后经加权赋值法综合分析获得6个候选基因的表达稳定性为ActinCyPAUbiEF1α18STubu,即Actin为表达最稳定的基因。因此,Actin可以作为衡量亚硝态氮胁迫后菲律宾蛤仔鳃组织中基因表达的内参基因。     

Diversity of antibiotic resistance genes and encoding ribosomal protection proteins gene in livestock waste polluted environment

The rapid development and increase of antibiotic resistance are global phenomena resulting from the extensive use of antibiotics in human clinics and animal feeding operations. Antibiotics can promote the occurrence of antibiotic resistance genes (ARGs), which can be transferred horizontally to humans and animals through water and the food chain. In this study, the presence and abundance of ARGs in livestock waste was monitored by quantitative PCR. A diverse set of bacteria and tetracycline resistance genes encoding ribosomal protection proteins (RPPs) from three livestock farms and a river were analyzed through denaturing gradient gel electrophoresis (DGGE). The abundance of sul(I) was 10³ to 10⁵ orders of magnitude higher than that of sul(II). Among 11 tet-ARGs, the most abundant was tet(O). The results regarding bacterial diversity indicated that the presence of antibiotics might have an evident impact on bacterial diversity at every site, particularly at the investigated swine producer. The effect of livestock waste on the bacterial diversity of soil was stronger than that of water. Furthermore, a sequencing analysis showed that tet(M) exhibited two genotypes, while the other RPPs-encoding genes exhibited at least three genotypes. This study showed that various ARGs and RPPs-encoding genes are particularly widespread among livestock.     

铜绿假单胞菌增强型绿色荧光蛋白标记的研究

根据增强型绿色荧光蛋白(EGFP)基因设计了上下游引物P1和P2,通过对铜绿假单胞菌菌株X3绿色荧光蛋白标记研究,构建了铜绿假单胞菌标记系统,获得带有EGFP标记的基因工程菌X9,为进行相关的跟踪研究创造了条件.该菌株绿色荧光标记性能稳定.通过转化子基因组DNAPCR和斑点杂交检测,外源EGFP基因存在于转化子染色体上. 图 6参 14     

生物强化系统微生物分子诊断技术的应用及新发展

现代分子生物技术的飞速发展及其在环境研究领域的应用，为生物强化技术的研究和发展提供了新方法和新思路。本文从生物强化系统特异微生物检测及定量化技术、生物强化系统微生物群落结构组成及动态演替规律研究、生物强化作用机制的分子生物学解析、生物强化菌的基因工程构建、生物强化系统微生物的安全释放及控制技术几方面，对生物强化系统微生物分子诊断技术的应用和发展作了较为全面的综述。     

火山岩填料曝气生物滤池的SNAD工艺启动特性及功能菌丰度演替

为研究同步亚硝化、厌氧氨氧化耦合反硝化(SNAD)工艺在浸没式生物滤池反应器(SBAF)内的运行特性,同时接种亚硝化污泥和富集ANAMMOX的填料启动SNAD反应器.结果表明在60 mg·L~(-1)有机物浓度下,自养脱氮和反硝化实现较好的耦合,并在该浓度下稳定运行了67 d,其总氮去除率最高可达92.0%,COD去除率最高达82.9%,最高总氮去除负荷为2.3 kg·(m~3·d)~(-1).与全程自养脱氮(CANON)工艺相比,SNAD工艺的平均总氮去除率提高了12.6%.荧光定量PCR结果显示,系统启动后AOB菌的丰度有所增长,ANAMMOX菌的丰度增长了1个数量级,而NOB菌和反硝化菌的数量维持在较低水平(小于10~7 copies·g~(-1)),表明以火山岩为填料的浸没式生物滤池反应器有利于ANAMMOX和AOB的协同生长,可快速实现SAND工艺的启动.     

抗生素抗性基因在两级废水处理系统中的分布和去除

废水处理系统被认为是水环境中抗生素抗性基因(ARGs)的重要污染源.为探究ARGs在废水处理系统中的分布特征和去除情况,选取某精细化工园区内的制药废水处理系统和园区综合性废水处理系统,使用PCR和实时荧光定量PCR对不同处理单元中ARGs的存在情况和丰度变化进行研究.在两个系统进水中分别检出了10种和15种ARGs,其中以四环素类和磺胺类ARGs居多,并首次检出了dfrA13基因.进水中sulⅠ和sulⅡ基因的丰度最高,随后依次是dfrA13、tetQ、floR、tetO和tetW基因.制药废水处理系统使总ARGs浓度上升了0.21个数量级,出水汇入园区综合性废水处理系统再次处理,其对综合性废水处理系统进水中总ARGs的贡献率为5.05%.综合性废水处理系统使总ARGs浓度下降了1.03个数量级,残留ARGs同最终出水一起直接排海,对近海环境中微生物群落的潜在影响有待深入研究.     

进水浓度或HRT变化时微好氧处理微生态机制研究

考察改变进水COD浓度（11 000、 15 000、 30 000 mg/Ｌ）或水力停留时间（HRT为42 h、 25 h）对模拟高浓度有机废水微好氧连续小试处理效果的影响,并采用荧光原位杂交-流式细胞术（FISH-FCM）、聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE) 等分子生物学技术及Biolog　FF微孔板法分析了不同处理阶段稳定期曝气柱污泥的微生态变化,以探讨进水COD浓度或HRT改变时微好氧处理效果变化的微生态机制.结果表明,在全部4个处理阶段,曝气柱污泥中酵母含量均保持在＞99.9％的水平.随着进水COD浓度的上升,污泥浓度由2.0 g/Ｌ上升到7.3 g/Ｌ,COD比去除速度由2.3 kg/（kg·d）下降到1.7 kg/（kg·d）,曝气柱污泥真菌群落结构多样性指数由2.05上升至2.19,代谢多样性指数由4.42上升至4.45;而随着HRT下降,污泥浓度从7.3 g/Ｌ下降至6.0 g/Ｌ,COD比去除速度从1.7 kg/（kg·d）上升至2.8 kg/（kg·d）,真菌群落结构多样性指数和代谢多样性指数则分别从2.19和4.45降至0.79和4.36.作为提高进水有机负荷的主要措施,提高进水COD浓度和缩短HRT对于废水处理效果和曝气柱微生态存在截然相反的影响.     

金鱼组织中壬基酚的含量检测新方法探讨

建立一种直接检测环境中痕量壬基酚的外切酶保护-荧光定量PCR方法。首先向含雌激素受体及相关蛋白的细胞溶质中加入壬基酚-丙酮溶液,使受体蛋白活化,从而在体外与含雌激素反应位点的双链结合DNA作用形成壬基酚-雌激素受体-DNA复合物。复合物用核酸外切酶和S1核酸酶消解,去除未受到蛋白质保护的结合DNA。将消解后产物作为模板,进行荧光定量PCR扩增反应,建立壬基酚浓度与标准DNA拷贝数的对数之间的线性关系为：y（壬基酚浓度的对数）=1.637x（标准DNA拷贝数的对数）-9.505。该法检出限为10^-8g/L,用该法对金鱼肝脏组织中壬基酚进行检测,添加回收率水平在98.5%112.2%,变异系数在4.3%以下。     

Do land utilization patterns a ect methanotrophic communities in a Chinese upland red soil?

Soil samples were collected from three plots under di erent land utilization patterns including degradation, farming, and restoration. The abundances of methanotrophs were quantified using real-time polymerase chain reaction (PCR) based on the pmoA and 16S rRNA genes, and the community fingerprint was analyzed using denaturing gradient gel electrophoresis (DGGE) aiming at pmoA gene. Significantly lower 16S rRNA and pmoA genes copies were found in the degradation treatment than in farming and restoration. Higher abundances of Type I than those of Type II methanotrophs were detected in all treatments. The treatment of farming was clearly separated from degradation and restoration according to the DGGE profile by cluster analysis. The lowest diversity indices were observed in the F (farming plot), suggesting that the community structure was strongly a ected by farming activities. There were significantly positive correlations between the copy numbers of pmoA also Type II-related 16S rRNA genes and soil available K content. Strong negative and positive correlations were found between Type I and soil pH, and available P content, respectively. We concluded that the vegetation cover or not, soil characteristics including pH and nutrients of P and K as a result of anthropogenic disturbance may be key factors a ecting methanotrophic communities in upland soil.