人X染色体间期细胞遗传学研究

应用人X染色体α卫星DNA探针进行X染色体正常或异常个体的外周血淋巴细胞染色体和间期核的原位杂交,在R显带的中期分裂相上,绝大部分杂交颂粒位于X染色体着丝粒区(p11→q11);在间期核内则显现与X染色体数相一致的银颗粒簇,其中相当部分位于核边缘区。实验结果表明,用原位杂交来检测X染色体数目,比记数Barr小体的方法可靠。本文还就α卫星DNA探针在间期细胞遗传学方面广泛的应用做了讨论。     

Effects of Vascular Endothelial Growth Factor 165 on Bone Tissue Engineering

To study the relationship between vascular endothelial growth factor (VEGF) and formation and repair of engineering bone, second-generation bone marrow stromal cells (BMSCs) of New Zealand white rabbits that were separated in vitro were transfected with VEGF 165 gene vectors by adenovirus to detect gene expressions. Transfected BMSCs and β-tricalcium phosphate material were complexed and implanted at the femoral injury sites of the study group (n = 12), and the control group (n = 12) were implanted with engineering bones that were not transfected with VEGF. Femoral recoveries of the two groups were observed on the 15th, 30th, 45th and 60th days, and their vascularization and ossification statuses were observed by immunohistochemical methods. The BMSCs transfected with VEGF highly expressed VEGF genes and excreted VEGF. The two groups both experienced increased vascularization and bone volume after implantation (t = 7.92, P<0.05), and the increases of the study group were significantly higher than those of the control group (t = 6.92, P<0.05). VEGF is clinically applicable because it can accelerate the formation and repair of engineering bone by promoting vascularization and ossification.     

Abundance and Diversity of Ammonia-Oxidizing Bacteria and Archaea in Cold Springs on the Qinghai-Tibet Plateau

The cold springs underlain by gas hydrates on the Qinghai-Tibet Plateau (QTP) are similar to deep-sea cold seeps with respect to methane biogeochemistry. Previous studies have shown that ammonia oxidizing bacteria (AOB) and archaea (AOA) are actively present and play important roles in the carbon/nitrogen cycles in cold seeps. Studying AOA and AOB communities in the QTP cold springs will be of great importance to our understanding of carbon and nitrogen cycling dynamics related to the underlying gas hydrates on the QTP. Thus, the abundance and diversity of AOB and AOA in sediments of four cold springs underlain by gas hydrates on the QTP were determined by using quantitative polymerase chain reaction and amoA gene (encoding ammonia monooxygenase involved in ammonia oxidation) phylogenetic analysis. The results showed that the AOB and AOA amoA gene abundances were at 10³–10⁴ copies per gram of the sediments in the investigated cold springs. The AOB population consisted of Nitrosospira and Nitrosomonas in contrast with the mere presence of Nitrosospira in marine cold seeps. The AOB diversity was higher in cold springs than in cold seeps. The AOA population was mainly composed of Nitrososphaera, in contrast with the dominance of Nitrosopumilus in cold seeps. The terrestrial origin and high level of dissolved oxygen of the cold springs may be the main factors accounting for the observed differences in AOB and AOA populations between the QTP cold springs and marine cold seeps.     

A new method for the determination of critical polyethylene glycol concentration for selective precipitation of DNA fragments

Separation strategies based on size-selective precipitation of DNA fragments with polyethylene glycol (PEG) have been used for achieving desired DNA interval in automated sample preparation for next-generation sequencing. By varying PEG concentration, DNA fragments of different sizes can be precipitated onto surfaces of carboxyl-coated paramagnetic particles selectively, and therefore, the desired DNA interval can be obtained. However, one of the crucial points in this approach is to determine the critical PEG concentration for DNA fragment of a certain size. The aim of this work was to develop a convenient and reliable method for accurately determining the critical PEG concentration. In our method, at a fixed concentration of sodium chloride (NaCl), recovered DNA samples obtained with different PEG concentrations were directly quantified, and their concentrations as a function of the PEG concentration were fitted by the logistic function. The critical PEG value was easily and accurately determined from the fitted logistic function. The repeatability and stability of the critical PEG value were assessed, showing an excellent reliability of the method. Based on this method, critical PEG values of different-size DNA fragments were determined at different NaCl concentrations. The effectiveness of the method was also demonstrated by selective precipitation of DNA fragments.     

毕赤酵母中tRNAPro CCG基因的表达及其作用效果

转运核糖核酸 (tRNA) 是蛋白质合成过程中重要参与成分之一,为了探索稀有密码子对应的tRNA (稀少tRNA) 丰度改变对外源基因表达量的影响,文中构建了毕赤酵母稀少tRNA基因与外源基因共表达体系。首先在GFP基因中添加由4个连续脯氨酸稀有密码子CCG组成的阻遏区,结果显示该GFP基因的表达量明显降低。然后将带有阻遏区的GFP基因和tRNAPro CCG基因顺次连接于pPIC9K载体上,在毕赤酵母GS115中共表达,结果使GFP表达量提高了4.9%;另将带有阻遏区的GFP基因和tRNAPro CCG基因分别连接于pPIC9K和pFLDα载体,在毕赤酵母GS115中共表达,GFP表达量最高提高了12.5%;应用同样方式将tRNAPro CCG基因与NFATc3T-GFP融合基因共表达,其表达量提高了21.3%。可见,tRNAPro CCG在毕赤酵母GS115中确为稀少tRNA,通过共表达tRNAPro CCG基因可显著提高带有连续该密码子的外源基因表达量,并且,文中构建的共表达体系将同样适用于其他稀少tRNA基因的筛选和验证。     

三峡库区水体中固氮微生物多样性及其影响因素

【目的】研究分析不同时空条件下三峡库区水体固氮微生物多样性,并探讨其与地球化学参数的相关性。【方法】采集三峡库区不同时间(三月份和六月份)和空间(干流与支流)的水体样品,对其进行地球化学参数分析,并通过构建克隆文库分析样品中固氮功能基因(nifH)的多样性进而探讨其与水体地化参数的相关关系。【结果】统计分析显示三峡库区水体固氮微生物α-多样性和群落组成具有时空差异。支流水体样品的固氮微生物α-多样性高于干流水体样品;六月水体样品的固氮微生物α-多样性高于三月水体样品。三峡库区三月水体样品中的固氮微生物群落以Proteobacteria (50.3%)和Firmicutes (40.0%)为主;六月水体样品的固氮微生物群落以Proteobacteria(48.4%)、Firmicutes(25.4%)和Cyanobacteria(19.0%)为主。Mantel检验结果显示:固氮微生物群落结构的差异与温度、pH和DIC等地球化学参数具有显著(P0.05)相关性,其中温度和pH的相关性系数最大。【结论】三峡库区固氮微生物的种群结构和多样性具有时空差异,影响三峡水库水体中固氮微生物群落结构与多样性的主要环境因素为温度和pH,同时浊度、DIC、氨氮也对库区水体固氮微生物群落结构和多样性有一定的影响。     

云南地区热泉中氨氧化菌丰度对环境条件的响应

【目的】研究热泉中的氨氧化菌对于理解全球氮循环作用至关重要,而人们对于热泉中环境条件对氨氧化菌丰度分布的影响还知之甚少。本研究旨在研究云南热泉中氨氧化菌的丰度以及热泉环境因子(例如:温度、氨浓度及pH等)对氨氧化菌丰度的影响。【方法】在所选取的热泉中,采集沉积物、菌席或泉华样品。使用RNA逆转录、定量聚合酶链式反应及荧光原位杂交等技术对样品中各微生物种群进行定量分析。【结果】所选取的热泉沉积物、菌席或泉华中微生物总量大约为108-109细胞/g。其中,氨氧化古菌(AOA)占样品中微生物总量的0.02-1.32%,而氨氧化细菌数量低于检测下限。地球化学参数和AOA相对丰度的相关性统计分析显示,氨氧化古菌相对丰度值与NH3、NO2-、NO3-浓度和温度等具有统计学意义上的相关性,而其与Fe2+和及盐度无统计学意义上的相关性。【结论】在所调查的热泉中,氨氧化微生物种群主要由AOA组成,AOA在热泉中的氨氧化生物地球化学过程中起着重要作用。热泉中多个环境因子一起控制着AOA丰度在不同热泉中的分布特征,而某些环境因子,如盐度-和Fe2+浓度,可能不是控制AOA分布特征的关键因素。     

Microbial response to salinity change in Lake Chaka, a hypersaline lake on Tibetan plateau

Previous investigations of the salinity effects on the microbial community composition have largely been limited to dynamic estuaries and coastal solar salterns. In this study, the effects of salinity and mineralogy on microbial community composition was studied by using a 900-cm sediment core collected from a stable, inland hypersaline lake, Lake Chaka, on the Tibetan Plateau, north-western China. This core, spanning a time of 17,000 years, was unique in that it possessed an entire range of salinity from freshwater clays and silty sands at the bottom to gypsum and glauberite in the middle, to halite at the top. Bacterial and archaeal communities were studied along the length of this core using an integrated approach combining mineralogy and geochemistry, molecular microbiology (16S rRNA gene analysis and quantitative polymerase chain reaction), cultivation and lipid biomarker analyses. Systematic changes in microbial community composition were correlated with the salinity gradient, but not with mineralogy. Bacterial community was dominated by the Firmicutes-related environmental sequences and known species (including sulfate-reducing bacteria) in the freshwater sediments at the bottom, but by halophilic and halotolerant Betaproteobacteria and Bacteroidetes in the hypersaline sediments at the top. Succession of proteobacterial groups along the salinity gradient, typically observed in free-living bacterial communities, was not observed in the sediment-associated community. Among Archaea, the Crenarchaeota were predominant in the bottom freshwater sediments, but the halophilic Halobacteriales of the Euryarchaeota was the most important group in the hypersaline sediments. Multiple isolates were obtained along the whole length of the core, and their salinity tolerance was consistent with the geochemical conditions. Iron-reducing bacteria were isolated in the freshwater sediments, which were capable of reducing structural Fe(III) in the Fe(III)-rich clay minerals predominant in the source sediment. These data have important implications for understanding how microorganisms respond to increased salinity in stable, inland water bodies.