天蓝苜蓿锌胁迫下实时定量PCR内参基因筛选

选取8个管家基因(h2a、act、18s、tub、ubi、ef1、cyp、gapdh)作为候选内参基因,以不同浓度Zn2+处理下接菌第30d的天蓝苜蓿(Medicago Lupulina L.)接种根为实验材料,筛选用于目的基因实时荧光定量PCR分析的最佳内参基因.研究表明:候选内参基因平均表达稳定性由高到低排序为:ef1>ubi>act>h2a>gapdh>cyp>18s>tub;在不同Zn2+浓度胁迫下,最适内参基因组合各不相同:Zn2+=100mg/kg时,最佳内参基因组合为tub (1.97)和ef1 (2.06); Zn2+=200mg/kg时,最佳内参基因组合为ef1 (1.41)和ubi (2.51); Zn2+=300mg/kg时,最佳内参基因组合为ef1 (1.41)和h2a (2.78); Zn2+=400mg/kg时,最佳内参基因组合为ef1 (2.45)和act (2.55).该研究可为重金属污染地天蓝苜蓿抗性基因和共生基因的表达分析提供理论依据.     

Research progress of nitrous oxide reductase gene (nosZII) and its relationship with the environment北大核心CSCD

The greenhouse effect has become increasingly serious globally. Nitrous oxide (N2O) is both the major ozone depleting substance and a potent greenhouse gas having a global warming potential 298 times that of CO2, and the N2O concentration is still increasing at an annual rate of about 0.8 × 10-9. Nitrous oxide reductase (N2OR) can reduce N2O to N2, and until recently, the nosZ gene was the only gene known to be able to encode N2OR. Besides the well-known nosZI, a new lineage of the N2O-reductase (nosZ clade II), which is abundant and widespread in soils, has been identified. In this paper, the main characters of nosZII-containing microbial communities and the related working mechanisms are summarized. In addition to the main differences between nosZI and nosZII, the important environmental factors that regulate the composition, abundance, and expression of nosZII-containing communities are also discussed in this paper. Studies have shown that nosZII communities are distributed among a diverse range of bacterial and archaeal phyla, such as Epsilon-proteobacteria, Bacteroidetes, and Aquificae. Interestingly, most of the nosZII microbes lack a nitrite reductase encoding gene (nirS or nirK) and are therefore unable to denitrify, indicating the importance of these communities as N2O sinks. Soil properties such as texture, pH, C/ N ratio, temperature, and lake physical gradient could regulate nosZII microbe abundance and diversity, and the pH and C/ N ratio may be the most important influencing factors. Studies on the ecological function of nosZII microbes have advanced considerably with the development of molecular biology technology. However, further studies on the community structure of nosZII microbes, the influencing factors of nosZII microbe abundance and diversity, and characteristics of nosZII strains with strong N2O reducing ability are needed. We hope to provide a theoretical basis that can be used to facilitate N2O reduction and relieve the greenhouse gas problem. © 2018 Science Press. All rights reserved.     

Cloning and expression of RING-finger E3 ubiquitin ligase CsSDIR1 gene in tea plant (Camellia sinensis)北大核心CSCD

Protein ubiquitination regulates many aspects of plant development and stress response. The RING-finger type E3 ubiquitin ligase SDIR1 (salt and drought induced ring finger 1) gene plays a key role in plant stress response. In this study, the full-length cDNA and the promoter sequences of CsSDIR1 were isolated from tea plants using the RT-PCR technology, and its bioinformatics characteristics were systematically analyzed. The expression patterns of CsSDIR1 in various tissues and in response to cold, drought, salt, and ABA treatments were also investigated by quantitative real-time RT-PCR (qRT-PCR). The sequence of CsSDIR1 contains a complete open reading frame of 831 bp, coding for a 276-long amino acid protein with a molecular weight of (Mr) 30.085 × 103 and a theoretical isoelectric point of 6.54. CsSDIR1 was predicted to be a hydrophobic protein localized on the intracellular membranes. The analysis of the amino acid sequence characteristics showed that CsSDIR1 contains two putative transmembrane domains at the N-terminus and a C3H2C3 RING-finger domain at the C-terminus; it shares high similarity with other plants' SDIR1, and has the closest relationship to Actinidia sinensis. A cis-acting regulatory elements prediction showed that the CsSDIR1 promoter contains many cis-acting elements, especially drought and salt stress response elements. The qRT-PCR analysis indicated that the CsSDIR1 gene has a high expression level in stems, followed by roots, leaves, and flowers; the expression of the CsSDIR1 gene is up-regulated by ABA, salt, and drought treatments, whereas it is down-regulated in response to cold stress. These results demonstrated that the CsSDIR1 gene might be involved in the plant stress response of tea trees. © 2018 Science Press. All rights reserved.     

丛枝菌根真菌砷酸盐还原酶基因RiarsC的克隆和功能分析

丛枝菌根真菌(AMF)在增强植物砷(As)抗性方面发挥着重要作用。已有相关研究表明,接种AMF能提高植物体内三价砷As(III)的比例,AMF可能参与了将五价砷As(V)还原为As(III)的过程从而提高了菌根植物的As抗性,但目前尚缺乏直接分子证据。本文从异形根孢囊霉(Rhizophagus irregularis)菌丝中克隆得到了一个砷酸盐还原酶基因RiarsC并进行序列分析。将该基因转入arsC缺陷型大肠杆菌(Escherichia coli)菌株WC3110(ΔarsC)中,通过As(V)抗性生长曲线和As形态测定,分析了该基因的功能。结果显示,RiarsC属于谷氧还蛋白-谷胱甘肽依赖的砷酸盐还原酶家族;RiarsC基因的表达显著提高了As敏感型E.coli菌株对As(V)的抗性,当培养基中As(V)浓度为100μmol·L-1时表现更加明显。As形态分析表明,表达RiarsC的E.coli菌株能够将培养基中71.03%的As(V)还原为As(III);与表达空载体的菌株相比,还原效率提高了61.98%。本研究证明了AMF的砷解毒还原能力,为进一步开展AMF的砷代谢机制研究提供了一定的分子生物学基础。     

CRISPR/Cas系统抵御细菌耐药

近年来,细菌耐药性问题在全球范围内日益严重,世界各地不断出现各种新型耐药基因及"超级细菌",以此形成的细菌耐药性污染已成为威胁全球公共卫生与环境安全的重大问题。目前除了在管理上规范合理用药和限制抗生素排放,应探讨抵御细菌耐药的分子机制,有效地从根本上遏制细菌耐药的产生。CRISPR系统作为一种天然免疫系统,可用来对抗入侵的外源性遗传物质,其结构和功能与细菌耐药及毒力因素密切相关,深入分析两者的关系有助于更好地理解细菌耐药机制,为防治细菌耐药提供了新的方向。     

胚胎期草甘膦铵盐暴露对中华鳖幼体的表型与生理表现的影响

草甘膦是全球范围内生产与使用量最大的除草剂。虽然草甘膦制剂被分于低毒等级,但是许多研究显示其对动物的生理、行为表达具有广泛的干扰作用。本研究将中华鳖胚胎暴露于不同浓度的草甘膦铵盐溶液以评估草甘膦除草剂对其胚胎发育及孵出幼体表型及生理表现的影响。结果显示:一定浓度范围(5～5 000 mg·L~(-1))内,草甘膦铵盐对中华鳖胚胎发育速率、孵出幼体大小、游泳能力、肝脏甘油三酯含量、超氧化物歧化酶活性、丙二醛含量以及热激蛋白70 mRNA相对表达量无显著影响。结果表明:一定浓度范围草甘膦除草剂暴露对中华鳖胚胎发育的影响较小,不改变孵出幼体的表型及其生理表现。     

甲基汞对大鼠脑即刻早期基因c-jun mRNA表达影响

为了探讨甲基汞神经毒性早期预报指标和信息传递分子机制,本文应用RT-PCR方法研究了不同浓度甲基汞暴露不同时间后,大鼠脑中即刻早期基因c-jun mRNA表达变化(对照组为0.9%生理盐水、暴露组浓度分别为0.05、0.5、5 mg·kg^-1;取样时间分别为20、60、240、1440 min).结果表明大鼠脑c-jun mRNA表达优先于汞的蓄积,大鼠脑c-jun mRNA表达变化对于甲基汞神经毒性具有早期预报作用, 即刻早期基因c-jun参与了甲基汞对中枢神经系统损害的毒性过程.     

Community diversity and distribution of ammonia-oxidizing archaea in marsh wetlands in the black soil zone in North-east China

AOA amoA genes in the soils of the two wetlands affiliated with three lineages. The main drivers of AOA community were pH and total organic carbon and ammonium. The soil characteristics rather than the vegetation control the AOA community. Since its first detection, ammonia-oxidizing archaea (AOA) have been proven to be ubiquitous in aquatic and terrestrial ecosystems. In this study, two freshwater marsh wetlands- the Honghe wetland and Qixinghe wetland – in the black soil zone in North-east China were chosen to investigate the AOA community diversity and distribution in wetland soils with different vegetation and depth. In the Honghe wetland, two sampling locations were chosen as the dominant plant transited from Deyeuxia to Carex. In the Qixinghe wetland, one sample location that was dominated by Deyeuxia was chosen. Samples of each location were collected from three different depths, and Illumina MiSeq platform was used to generate the AOA amoA gene archive. The results showed that the AOA amoA genes in the soils of the two wetlands were affiliated with three lineages: Nitrososphaera, Nitrosotalea, and Nitrosopumilus clusters. The different dominant status of these AOA lineages indicated their differences in adapting to acidic habitat, oxygenic/hypoxic alternation, organic matter, and other environmental factors, suggesting high diversity among AOA in marsh soils. The main driver of the AOA community was pH, along with organic carbon and ammonium nitrogen, which also played an important role combined with many other environmental factors. Thus, soil physiochemical characteristics, rather than vegetation, were the main cause of AOA community diversity in the wetlands in the black soil zone in China.     

16S rRNA基因技术在环境科学领域中的应用

随着分子生物学的发展，16S rRNA基因技术被逐渐应用到环境科学领域中。目前在环境保护和治理中，该技术主要被用于鉴定污染物的生物降解菌和分析环境样品中的微生物群落多样性，由于它不必将微生物培养分离出来，也就避免了在培养过程中可能出现的微生物去失的情况。本文对16S rRNA基因技术及其在环境科学领域中的应用现状和发展作了一简要介绍，并对16S rRNA基因技术存在的不足进行了讨论。