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.     

Evaluating the outcomes of genetic rescue attempts

Augmenting gene flow is a powerful tool for the conservation of small, isolated populations. However, genetic rescue attempts have largely been limited to populations at the brink of extinction, in part due to concerns over negative outcomes (e.g., outbreeding depression). Increasing habitat fragmentation may necessitate more proactive genetic management. Broader application of augmented gene flow will, in turn, require rigorous evaluation to increase confidence and identify pitfalls in this approach. To date, there has been no assessment of best monitoring practices for genetic rescue attempts. We used genomically explicit, individual-based simulations to examine the effectiveness of common approaches (i.e., tests for increases in fitness, migrant ancestry, heterozygosity, and abundance) for determining whether genetic rescue or outbreeding depression occurred. Statistical power to detect the effects of gene flow on fitness was high (≥0.8) when effect sizes were large, a finding consistent with those from previous studies on severely inbred populations. However, smaller effects of gene flow on fitness can appreciably affect persistence probability but current evaluation approaches fail to provide results from which reliable inferences can be drawn. The power of the metrics we examined to evaluate genetic rescue attempts depended on the time since gene flow and whether gene flow was beneficial or deleterious. Encouragingly, the use of multiple metrics provided nonredundant information and improved inference reliability, highlighting the importance of intensive monitoring efforts. Further development of best practices for evaluating genetic rescue attempts will be crucial for a responsible transition to increased use of translocations to decrease extinction risk.     

Phenological mismatch and the effectiveness of assisted gene flow

The persistence of narrowly adapted species under climate change will depend on their ability to migrate apace with their historical climatic envelope or to adapt in place to maintain fitness. This second path to persistence can only occur if there is sufficient genetic variance for response to new selection regimes. Inadequate levels of genetic variation can be remedied through assisted gene flow (AGF), that is the intentional introduction of individuals genetically adapted to localities with historic climates similar to the current or future climate experienced by the resident population. However, the timing of reproduction is frequently adapted to local conditions. Phenological mismatch between residents and migrants can reduce resident × migrant mating frequencies, slowing the introgression of migrant alleles into the resident genetic background and impeding evolutionary rescue efforts. Focusing on plants, we devised a method to estimate the frequency of resident × migrant matings based on flowering schedules and applied it in an experiment that mimicked the first generation of an AGF program with Chamaecrista fasciculata, a prairie annual, under current and expected future temperature regimes. Phenological mismatch reduced the potential for resident × migrant matings by 40–90%, regardless of thermal treatment. The most successful migrant sires were the most resident like in their flowering time, further biasing the genetic admixture between resident and migrant populations. Other loci contributing to local adaptation—heat‐tolerance genes, for instance—may be in linkage disequilibrium with phenology when residents and migrants are combined into a single mating pool. Thus, introgression of potentially adaptive migrant alleles into the resident genetic background is slowed when selection acts against migrant phenology. Successful AGF programs may require sustained high immigration rates or preliminary breeding programs when phenologically matched migrant source populations are unavailable.     

土壤质地对自养固碳微生物及其同化碳的影响

自养微生物可同化大气中的CO2并将其转化为土壤有机碳,对提高农田土壤的碳吸收和碳储存有重要意义,然而土壤质地对自养固碳微生物功能种群及其同化碳的影响机制还不清楚.本研究选取亚热带地区同一母质发育而成的两种质地水稻土壤(壤质黏土和砂质黏壤土),通过14C-CO2连续标记技术结合室内模拟培养实验,探讨土壤质地对自养微生物同化碳(14C-SOC)、自养微生物截留碳(14C-MBC)和自养微生物可溶性碳(14C-DOC)的影响.以固碳功能基因(cbb L基因)作为指示基因,结合PCR和克隆测序技术,分析不同质地土壤自养固碳微生物群落结构和多样性的差异.结果表明,壤质黏土14C-SOC、14C-MBC和14C-DOC平均含量分别为133.81、40.16和8.10 mg·kg-1,均显著高于砂质黏壤土14C-SOC(104.95 mg·kg-1)、14C-MBC(33.26 mg·kg-1)和14C-DOC(4.18 mg·kg-1)平均含量(P0.05),说明土壤质地显著影响了土壤自养微生物碳同化量以及自养微生物同化碳在土壤中的转化.稀疏曲线、细菌cbb L基因文库覆盖度以及多样性指数分析结果显示壤质黏土固碳细菌群落多样性高于砂质黏壤土.系统发育分析表明,壤质黏土细菌cbb L基因序列与Rhodoblastus acidophilus、Blastochloris viridis、Thauera humireducens、Mehylibium sp.、Variovorax sp.等具有一定的同源性,而砂质黏壤土cbb L基因序列主要与根瘤菌和放线菌同源.可见,土壤质地对自养固碳微生物群落结构和多样性产生了深刻的影响,壤质黏土中较高的黏粒含量、土壤养分含量和阳离子交换量可能有利于维持更高的自养固碳微生物多样性和活性,从而导致不同质地土壤自养微生物碳同化量及其转化存在显著差异.     

基于受体作用理论的类雌激素效应测试方法

以17β雌二醇为标准参考物质,对基于受体作用理论的环境激素分析方法实验条件进行了重构和优化,建立了环境类雌激素分析方法。结果表明:用营养缺陷型培养基30℃恒温振荡66 h培养时间进行重组基因酵母的复苏与增殖,20℃～35℃水浴样品与酵母混合液培养3～6 h暴露,优先选用机溶剂处理法释放β-半乳糖苷酶,35℃恒温水浴进行酶活测试反应,采用反应终止后2.5 h 420 nm波长进行分析测试,同时实际样品的分析,加测不少于一个的稀释水样效应值,可使方法更加科学高效、准确度高,应用于各类实际样品分析。     

温度分化对APBR反应器性能及产甲烷菌群落的影响

为了研究温度分化对固定床厌氧反应器(anaerobic packed bed reactor,APBR)牛粪发酵处理效果及产甲烷菌群落的影响,反应器发酵温度从室温(22℃±1℃)阶梯式分化到低温(15℃±1℃)、中温(37℃±1℃)和高温(55℃±1℃).温度变化的过程中,温度越高COD(chemical oxygen demand)去除率和日总产气量越高,分化后COD去除率分别为25%、45%、60%,相应的日产气量为2.3、4.0、8.5 L·d-1,但是甲烷含量基本保持不变(~60%);温度突然变化造成挥发性脂肪酸含量骤然增加,并处于波动状态.16S r RNA基因克隆文库法分析表明,室温时包含广古菌门中的常见重要产甲烷菌MBT(甲烷杆菌目)、Mst(甲烷鬃菌科)、Msc(甲烷八叠球菌科)和MMB(甲烷微菌目),以及嗜热菌,也有少部分泉古生菌门,发酵温度分化后,产甲烷菌多样性减少,中温条件下产甲烷菌种类相对较少.定量PCR表明Mst、MMB和Msc总基因浓度都有所减少,并且温度越高减少越多,各菌数量相对比例变化较大,但Mst仍为优势产甲烷菌.     

曝气灌溉条件下土壤N2O排放特征及影响因子分析

为了明确曝气灌溉下土壤N₂O排放特征及主要影响因子,实验设置了2个灌水量（70%和90%田间持水量）和2个增氧水平（5,40mg/L）,采用静态箱法和qPCR技术对土壤N₂O通量及土壤关键功能基因进行测定,研究不同灌水量和增氧水平对土壤充水孔隙度、溶解氧、氧化还原电位（Eh）、矿质氮及氨氧化古菌（AOA）、氨氧化细菌（AOB）和反硝化基因（narG和nosZ）的影响.结果表明：培养过程中,各处理N₂O排放通量均呈现先增加后降低的趋势,于灌溉后1d达到峰值;曝气量和灌水量的增加可显著增加土壤N₂O的排放通量和排放峰值.灌溉造成土壤含水量增加的同时,降低了土壤溶解氧和Eh;曝气可提高土壤溶解氧和Eh,改善土壤通气性（P<0.05）,而对土壤充水孔隙度无显著影响.土壤充水孔隙度、Eh、NO₃^--N含量是曝气灌溉下驱动土壤N₂O排放的主要理化因子.曝气显著增加了AOA的基因拷贝数,且N₂O排放与AOA的基因拷贝数呈显著正相关关系（P<0.05）.研究结果为进一步明确曝气灌溉对土壤N₂O排放的影响机制和曝气灌溉模式下农田N₂O排放管理提供支撑.     

低浓度环丙沙星对曝气生物滤池生物膜硝化过程及硝化微生物的作用影响

以曝气生物滤池(BAF)作为研究对象,考察了低浓度环丙沙星(CIP)对其生物膜微生物硝化过程及其功能微生物的作用影响,采用荧光定量PCR方法定量检测分析了4种环丙沙星抗性基因(CIP-ARGs)在硝化作用过程中的丰度变化,并探讨了其与硝化微生物之间的相关关系.结果表明,CIP对生物膜氨氧化阶段影响较小,但对亚硝酸盐氧化阶段具有一定抑制作用.通过对氨氧化细菌(AOB)和亚硝酸盐氧化细菌(NOB,包括Nitrobacter和Nitrospira)的定量检测结果分析可知,CIP对生物膜亚硝酸盐氧化转化过程的抑制主要是通过对Nitrobacter和Nitrospira的抑制实现的.此外,CIP的加入对生物膜中aac和qepA抗性基因的变化影响较小,但对parC和oqxB的影响较大.通过相关性分析可知,Nitrobacter与parC之间具有显著相关性,Nitrospira与oqxB之间同样具有显著相关性,推测生物膜中不同硝化微生物的遗传因子中可能携带有CIP-ARGs.     

基于hzsB功能基因研究典型湿地沉积物中厌氧氨氧化细菌的群落结构

采用基于厌氧氨氧化细菌功能基因hzs B(联氨合成酶关键基因)PCR扩增的高通量测序,研究了典型湿地沉积物中厌氧氨氧化细菌的群落结构.结果表明,沿纬度梯度分布的10个典型湿地沉积物样品的测序序列经处理后共得到190578条序列,在90%的相似度下聚类得到77个OTUs.基因丰度值显示,10个样品的基因拷贝数在5.42×10~4~4.20×10~6copies·g~(-1)之间.群落组成结果表明,在典型湿地沉积物中,序列主要隶属于‘Ca.Brocadia’(54.09%)、‘Ca.Jettenia’(30.97%)、‘Ca.Kuenenia’(6.95%)、‘Ca.Scalindua’(7.91%).相对丰度结果显示,各样点的主导菌属有差别.多样性分析(OTUs水平)表明,在本研究的湿地沉积物中,低纬度样点的多样性普遍高于高纬度样点.PCo A分析(OTUs水平)表明,各湿地样点厌氧氨氧化细菌的群落组成表现出明显的差异性.相关性分析(α=0.10)表明,在典型湿地沉积物中,厌氧氨氧化细菌的丰度与有机质、总氮、总碳、总硫呈负相关.冗余分析表明,‘Ca.Jettenia’与NH_4~+显著正相关,‘Ca.Scalindua’与盐度显著正相关,‘Ca.Kuenenia’在所有测定理化因子中与盐度的正相关性最显著.综上,针对厌氧氨氧化细菌hzs B功能基因扩增进行高通量测序较好地揭示了典型湿地沉积物中厌氧氨氧化细菌的多样性、群落组成及其与环境因子的关系.