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Grassland degradation has become a worldwide ecological problem. Although soil microorganisms, as the main participants in the process of grassland degradation, play a key role in maintaining ecosystem function and improving soil productivity, little is known about the changes in microbial communities caused by grassland degradation and their relationship with soil properties and plant communities. In this study, we used Illumina MiSeq sequencing to analyze the soil fungal communities of subalpine meadow soil at four different degradation stages[i.e., non-degraded (ND), lightly degraded (LD), moderately degraded (MD), and heavily degraded (HD)] on Mount Wutai. The results showed that Ascomycota, Basidiomycota, and Zygomycota were the dominant phyla of soil fungi in the subalpine meadow, regardless of degradation stage. LEfSe showed that the subalpine meadows with different degradation degrees were enriched with different biomarkers. Compared with ND, MD and HD were enriched with more pathogenic fungi. Moreover, HD apparently decreased the richness and Shannon indexes of soil fungal communities compared with those of ND. Non-metric multidimensional scaling (NMDS) and similarity analysis (ANOSIM) indicated that the compositions and structures of fungal communities were significantly different among meadows with different degradation degrees (

Background Pediatric enteritis is one of the infectious diseases in the digestive system that causes a variety of digestive problems, including diarrhea, vomiting, and bellyache in children. Clinically, Helicobacter pylori (H. pylori) infection is one of the common factors to cause pediatric enteritis. It has been demonstrated that aberrant expression of microRNAs (miRNAs) is found in gastrointestinal diseases caused by H. pylori, and we discovered a significant increase of miR-32-5p in H. pylori-related pediatric enteritis. However, the exact role of miR-32-5p in it is still unknown. Aim To investigate the role of aberrant miR-32-5p in pediatric enteritis induced by H. pylori. Methods MiR-32-5p expression was detected by quantitative real time-polymerase chain reaction. The biological role of miR-32-5p in H. pylori-treated intestinal epithelial cells was evaluated by Cell Counting Kit-8 assay and flow cytometry. The potential target of miR-32-5p was predicted with TargetScanHuman and verified by luciferase assay. The downstream mechanism of miR-32-5p was explored by using molecular biology methods. Results We found that miR-32-5p was overexpressed in serum of H. pylori-induced pediatric enteritis. Further investigation revealed that H. pylori infection promoted the death of intestinal epithelial cells, and increased miR-32-5p expression. Moreover, miR-32-5p mimic further facilitated apoptosis and inflammatory cytokine secretion of intestinal epithelial cells. Further exploration revealed that SMAD family member 6 (SMAD6) was the direct target of miR-32-5p, and SMAD6 overexpression partially rescued cell damage induced by H. pylori. The following experiments showed that miR-32-5p/SMAD6 participated in the apoptosis of intestinal epithelial cells induced by transforming growth factor-β-activated kinase 1 (TAK1)-p38 activation under H. pylori infection. Conclusion Our work uncovered the crucial role of aberrant expression of miR-32-5p in H. pylori-related pediatric enteritis, and suggested that the TAK1-p38 pathway is involved in it.

To investigate the molecular mechanism in stable cell strains expressing Mini-hF9 gene with nonsense mutation.Mini-hF9 gene and its nonsense mutants were transfected into HeLa cells independently, and stable cell strains were obtained after G418 resistance screening and monoclonal transformation. The altered splicing and protein expression of mRNA in Mini-hF9 gene in stable cell strains were detected by using RT-PCR and Western blot.The wild type and nonsense mutated human coagulation factor IX stable cell strains were constructed successfully, which were named HeLa-F9-WT, HeLa-F9-M1 and HeLa-F9-M2. Only normal splicing Norm was detected in the wild-type cell strain HeLa-F9-WT; Norm and Alt-S1 splicing were detected in HeLa-F9-M1; while Norm, Alt-S1 and Alt-S2 splicing were detected in HeLa-F9-M2.The nonsense associated altered splicing (NAS) pathway, which generated alternately spliced transcripts, might be triggered in coagulation factor IX gene with nonsense mutation.无义突变的人凝血因子IX小基因稳定细胞株选择性剪接的研究.探讨无义突变的人凝血因子IX小基因稳定细胞株发生选择性剪接的分子机制.将人凝血因子IX小基因（Mini-hF9）及其无义突变体分别转染哺乳动物细胞HeLa，经G418抗性筛选、单克隆化得到稳定细胞株；通过RT-PCR及Western blot分别检测稳定细胞株中Mini-hF9基因mRNA的选择性剪接及蛋白表达情况.成功构建了野生型和无义突变的人凝血因子IX小基因稳定细胞株HeLa-F9-WT、HeLa-F9-M1和HeLa-F9-M2。野生型稳定细胞株HeLa-F9-WT中扩增到正常剪接产物Norm；无义突变稳定细胞株HeLa-F9-M1中扩增到Norm和Alt-S1这两种剪接产物，HeLa-F9-M2中扩增到Norm、Alt-S1和Alt-S2这三种剪接产物.无义突变的凝血因子IX基因可能触发无义相关的选择性剪接（NAS）途径，导致选择性剪接的产生.

There are complex interrelationships between plant microorganisms (phyllosphere and rhizosphere) and host plants, which can promote plant growth and enhance the tolerance of host plants to stress. In this study, we selected the dominant species

Human activities and climate change cause the degradation of subalpine lake ecosystems, which induce the shift of microbial community structure. The spatio-temporal dynamics and the diversity maintenance mechanisms of bacterial communities in Gonghai Lake in Ningwu, Shanxi, were investigated by using Q-PCR and DGGE. The results showed that the temperature), pH, dissolved oxygen, electrical conductivity salinity, and ammonium nitrogen (NH

The composition and diversity of fungal communities are essential to maintain the ecosystem balance of subalpine lakes. The aquatic fungal communities at different depths from the subalpine Pipahai (PPH, 0, 2, 4 m), Mayinghai (MYH, 0, 2, 4, 6 m), and Gonghai (GH, 0, 2, 4, 6, 8 m) lakes were studied. In addition to that, the distribution pattern and diversity maintenance mechanism (determination process vs. random process) of fungal communities were explored using high-throughput sequencing. The results showed that the physicochemical parameters of the water were significantly different among the three lakes. The pH, electrical conductivity (EC), ammonia nitrogen (NH

BACKGROUND Pediatric enteritis is one of the infectious diseases in the digestive system that causes a variety of digestive problems, including diarrhea, vomiting, and bellyache in children. Clinically, Helicobacter pylori (H. pylori) infection is one of the common factors to cause pediatric enteritis. It has been demonstrated that aberrant expression of microRNAs (miRNAs) is found in gastrointestinal diseases caused by H. pylori, and we discovered a significant increase of miR-32-5p in H. pylori-related pediatric enteritis. However, the exact role of miR-32-5p in it is still unknown. AIM To investigate the role of aberrant miR-32-5p in pediatric enteritis induced by H. pylori. METHODS MiR-32-5p expression was detected by quantitative real time-polymerase chain reaction. The biological role of miR-32-5p in H. pylori-treated intestinal epithelial cells was evaluated by Cell Counting Kit-8 assay and flow cytometry. The potential target of miR-32-5p was predicted with TargetScanHuman and verified by luciferase assay. The downstream mechanism of miR-32-5p was explored by using molecular biology methods. RESULTS We found that miR-32-5p was overexpressed in serum of H. pylori-induced pediatric enteritis. Further investigation revealed that H. pylori infection promoted the death of intestinal epithelial cells, and increased miR-32-5p expression. Moreover, miR-32-5p mimic further facilitated apoptosis and inflammatory cytokine secretion of intestinal epithelial cells. Further exploration revealed that SMAD family member 6 (SMAD6) was the direct target of miR-32-5p, and SMAD6 overexpression partially rescued cell damage induced by H. pylori. The following experiments showed that miR-32-5p/SMAD6 participated in the apoptosis of intestinal epithelial cells induced by transforming growth factor-β-activated kinase 1 (TAK1)-p38 activation under H. pylori infection. CONCLUSION Our work uncovered the crucial role of aberrant expression of miR-32-5p in H. pylori–related pediatric enteritis, and suggested that the TAK1-p38 pathway is involved in it.

The maintaining mechanism of community diversity is the core of community ecology. The mine tailing is a good field for studying on the underlying mechanism of community diversity, as a kind of original bare land with heavy metal pollution, where the physicochemical characteristics of soil change with the restoration periods. We examined the driving forces for bacterial community diversity based on the investigation of edaphic factors, plant community, and bacterial communities in Shibahe copper mine tailing, Shanxi. The results showed that nutrient contents in soil increased with restoration periods. The seasonal dynamics of soil nutrient in different restoration time were different. Shannon diversity and richness of bacterial community showed an increasing trend, indicating community stability was improved with restoration. Influenced by plant community, the seasonal changes of those indices differed with restoration. Results from the RDA analysis showed that the diversity and structure of bacterial communities were determined by environmental factors (edaphic, plant and heavy metals). Results from the structure equation models further confirmed that soil nutrients (TC, TN, NO群落多样性的维持机制是群落生态学研究的热点问题.尾矿库作为人工原生裸地,土壤重金属含量较高,随着恢复年限的延长,土壤理化性质发生变化,是研究土壤微生物群落多样性驱动机制的理想场地.本研究在调查十八河尾矿库土壤因子、植物群落多样性和土壤细菌群落多样性的基础上,探究了局域小尺度下细菌群落结构和多样性的驱动机制.结果表明: 尾矿库土壤养分含量随恢复年限的延长显著提高,并具有一定的季节变化,不同恢复年限土壤养分含量的季节动态存在差异.细菌群落的Shannon多样性和丰富度指数随恢复年限呈显著增加趋势,群落稳定性逐步提高;而其季节变化受到植物群落的影响,在不同恢复年限下具有一定的差异.RDA分析显示,环境因子对土壤细菌群落多样性有决定作用.结构方程模型分析表明,土壤养分(TC、TN、NO

The relative importance of the deterministic versus stochastic processes underlying community dynamics has long been a central theme in community ecology, and is intensively debated in the field. Microbial communities play key roles in nutrient cycling and the flow of energy in ecosystems. The research on the structural dynamics of microbial community will provide data and theoretical support for understanding the assembly mechanisms of community, and for predicting the dynamics of microbial community under environmental stress. In this study, the Illumina MiSeq method was applied to investigate the structural dynamics of bacterial and fungal community in a dam of Shibahe mine tailings at different restoration stages (1-45 years). The results indicated that the soil physicochemical properties in the dam of mine tailings formed an ecological gradient, and the plant community showed succession along the restoration time. The diversity of plant communities was significantly correlated with soil nutrient contents but not with soil heavy metal contents. The structure of the microbial communities showed significant differences at different restoration stages of the dam land, in which Proteobacteria, Actinobacteria, Firmicutes, and Acidobacteria were the dominant bacterial phyla, and Ascomycota, Basidiomycota, and Zygomycota were the dominant fungal phyla. The assembly of the microbial community was shaped mainly by the soil nutrients and soil heavy metal contents, but plant diversity had no significant effect on the microbial community structure. It was suggested that edaphic factors drive the dynamics of microbial communities under the stress conditions of pH and heavy metals on small, local scales.

In order to reveal the effects of heavy metal pollution on microbial community compositions and microbial community diversity in tailing area,we conducted an experiment by examining the microbial community in tailing water,sediments and tailing sand in Shibahe copper tailing in Zhongtiao Mountain.Differences in microbial community compositions in different habitats and their relationships with environmental parameters were analyzed.The results showed that the richness and diversity of microbial community were the largest in tailing sand,but the lowest in tailing water.Microbial community compositions were similar between tailing water and sediments.There were significant positive correlations between the relative abundance of the dominant family (Sphingomonadaceae) and contents of heavy metals (Cd,Cu,Mn,Ni,Pb,Zn),while there were significant negative correlations between relative abundances of aulobacteraceae, Methylobacteriaceae, Nocardioidaceae, Microbacteriaceae, Micrococcaceae, Streptococcaceae and Paenibacillaceae and heavy metal contents.It showed that most of the bacteria were inhibited by heavy metals,but Sphingomonadaceae had a higher tolerance to heavy metals which may indicate that it has a potential for remediation of heavy metal contamination.

Soil microorganisms drive the biogeochemical process of carbon, nitrogen, phosphorus and sulfur, and play a key role in maintaining soil carbon sink and ecosystem function. The study on effects of environmental and spatial factors on the structure of microbial community in boreal coniferous forest soil will provide theoretical basis for making management measures in local forest ecosystem. Our research analyzed five soil fungi communities (LpMC1, LpMC2, PwMC, PtMC, and BMC) in four forest types, including

Environmental selection and dispersal limitation are two basic processes underlying community assembly. The relative importance of those two processes differs across scales, community identities, and community types. The processes responsible for structuring microbial communities in soil of temperate subalpine forest are poorly understood. Here, we investigated the relationship between soil bacterial community structure and environmental factors, and quantified the relative role of edaphic factors, vegetation, and spatial variables in shaping the structure of six soil bacterial communities (LpMC1, LpMC2, PwMC, PmMC, PtMC, and BMC) in five forest types including Larix principis-rupprechtii, Picea wilsonii, Picea meyeri, Pinus tabulaeformis, and Betula platyphylla in Pangquangou Nature Reserve by using PCR-DGGE technology. Our results showed that the structure and biodiversity of bacterial communities were significantly different among six communities. The biodiversity of bacterial community were higher in LpMC2 and PtMC, lowest in PmMC, and highest in LpMC1. Soil environmental factors, such as pH, soil water content, total carbon, total nitrogen, soil organic matter, available phosphorous, and soil enzymes, were significantly correlated with biodiversity and structure of soil bacterial community. The beta diversity of bacterial communities were significantly correlated with geographic distance, indicating the influence of dispersal limitation on the structure of bacterial community. The order of driving force on the structure of bacterial community was edaphic factors (0.27), spatial factor (0.19) and vegetation (0.15) in six samples. Using regional soil microbes from 10 samples around reserve as source community, results from the microcosm experiments showed that the edaphic factors were the predominant driving factors (0.35) on structure of artificial dispersal bacterial community, while the high diversity of source microbial community affected the structure of microcosm soil. In summary, at local scale, environmental selection predominantly determined the structural and biodiversity of soil bacterial communities in temperate subalpine forest, while dispersal limitation played a significant role. Such a result indicated that deterministic processes and stochastic processes played important roles in shaping the structure of soil bacterial community at local scale, with the former having the leading role. The composition of dispersal soil bacteria community was source-dependent but also modulated by local environmental selection.环境选择和扩散限制是生态系统中生物群落构建的两个基本过程,而两者相对作用的大小因研究尺度、群落属性和类型等有所不同.目前对温带亚高山森林土壤微生物群落构建的驱动因子和机制尚缺乏了解.本文利用PCR-DGGE技术研究庞泉沟自然保护区内5种典型森林包括华北落叶松林、青杄林、白杄林、油松林以及桦树林的6个森林土壤细菌群落(LpMC1、LpMC2、PwMC、PmMC、PtMC、BMC)的结构特征及其影响因素,分析细菌群落结构与环境因子的相关性,以及土壤因子、植被和空间因素对细菌群落结构的影响.结果表明: 研究区各样地土壤细菌群落的结构和生物多样性具有显著差异,低海拔落叶松和油松土壤细菌群落多样性较高(20条带),白杄林土壤细菌群落(13条带)多样性最低,高海拔落叶松土壤细菌群落多样性最高;土壤环境因子,如pH、土壤含水量、总碳、总氮、土壤有机质、速效磷以及土壤酶活性与土壤细菌群落多样性和结构显著相关;样地土壤细菌群落的beta多样性与群落的空间距离呈显著相关,表明扩散限制对群落结构具有一定的影响;方差分解分析结果显示,6个样地细菌群落结构的驱动因素大小依次为土壤因子(0.27)、空间因素(0.19)和植被(0.15);将区域土壤微生物作为“源群落”,微宇宙试验结果显示,土壤因子是细菌群落结构形成的主要驱动力(0.35),同时源群落丰富的物种多样性对微宇宙土壤细菌群落结构具有显著影响.总之,在局域尺度下,环境选择对温带森林土壤细菌群落结构动态和多样性发挥主导作用,地理距离对群落结构具有显著影响,即确定性过程和随机过程共同决定局域森林土壤细菌群落结构,前者占主导地位.对于土壤细菌群落而言,扩散群落的组成和结构受到源群落的多样性特征和环境因子的双重影响.

The complete chloroplast (cp) genome of Caragana microphylla (Fabaceae) has been characterized from Illumina pair-end sequencing. The whole cp genome is relatively short (130,029 bp) because it lacks an inverted repeat (IR) region. It comprises 110 genes, including four unique rRNAs, 30 tRNAs, and 76 protein-coding genes. Similar to other closely related plastomes, rpl22 and rps16 are absent. The overall GC content of C. microphylla cp genome is 34.3 %. The phylogenetic analysis suggested that C. microphylla is closely related to the genera of Astragalus.

Eukaryotic translation termination is governed by eRF1 and eRF3. eRF1 recognizes the stop codons and then hydrolyzes peptidyl-tRNA. eRF3, which facilitates the termination process, belongs to the GTPase superfamily. In this study, the effect of the MC domain of eRF1a (eRF1aMC) on the GTPase activity of eRF3 was analyzed using fluorescence spectra and high-performance liquid chromatography. The results indicated eRF1aMC promotes the GTPase activity of eRF3, which is similar to the role of eRF1a. Furthermore, the increased affinity of eRF3 for GTP induced by eRF1aMC was dependent on the concentration of Mg(2+). Changes in the secondary structure of eRF3C after binding GTP/GDP were detected by CD spectroscopy. The results revealed changes of conformation during formation of the eRF3C·GTP complex that were detected in the presence of eRF1a or eRF1aMC. The conformations of the eRF3C·eRF1a·GTP and eRF3C·eRF1aMC·GTP complexes were further altered upon the addition of Mg(2+). By contrast, there was no change in the conformation of GTP bound to free eRF3C or the eRF3C·eRF1aN complex. These results suggest that alterations in the conformation of GTP bound to eRF3 is dependent on eRF1a and Mg(2+), whereas the MC domain of eRF1a is responsible for the change in the conformation of GTP bound to eRF3 in Euplotes octocarinatus.

This study was purposed to construct the recombinant hF9 minigene and its stable nonsense mutant cell lines, and to investigate its significance. Minigene hF9 was cloned into the mammalian expression vector pCMV-Tag3B; a nonsense mutant containing a premature termination codon (PTC) in the 121(st) amino acid residue was obtained by PCR site-directed mutagenesis; minigene hF9 and nonsense mutant were respectively transfected into HepG2 cells with G418 treatment to get stable HepG2-WT and HepG2-N cell lines. The results confirmed that the minigene hF9 and nonsense mutant were constructed successfully. The gene of interest was amplified by RT-PCR from the stable cell lines, and the minigene hF9 was expressed in the stable cell lines. It is concluded that the recombinant hF9 minigene and its stable nonsense mutant cell lines are constructed successfully. The cell lines can be used to screen the drugs treating the nonsense mutation-caused hemophilia according to PTC read-through approaches.

In eukaryotes, eRF3 participates in translation termination and belongs to the superfamily of GTPases. In this work, the dissociation constants for nucleosides bound to Euplotes octocarinatus eRF3 in the presence and absence of eRF1a were determined using fluorescence spectra methods. Furthermore, a GTP hydrolyzing assay of eRF3 was carried out using an HPLC method, and the kinetic parameters for GTP hydrolysis by eRF3 were determined. Consistent with data from humans, the results showed that eRF1a promoted the binding of GTP to eRF3 and the GTP hydrolyzing activity of eRF3. However, in contrast to the lack of GTP binding in the absence of eRF1 in human eRF3, the E. octocarinatus eRF3 was able to bind GTP by itself. The nucleotide binding affinity of the E. octocarinatus eRF3 also differed from the human data. A structure model and amino acid sequence alignment of potential G domains indicated that these differences may be due to valine 317 and glutamate 452 displacing the conserved glycine and lysine involved in GTP binding.

The purpose of this study was to construct 4 types of nonsense-mutated eukaryotic expression plasmids of fIX gene, using pcDNA3.1 plasmid containing fIX cDNA as template, and to identify, then to perform their expression in COS-7 cells. These stop mutants constructed by site-directed mutagenesis based on PCR, and further confirmed by DNA sequencing. COS-7 cells were transfected with either the wild-type or mutated fIX expression constructs, then the relative expression levels of fIX mRNA were detected by real time fluorescent quantitative PCR. The result showed that except the designed sites, there were no other nucleotide mutation in the sequences of four nonsense mutants. The results of real time PCR proved that the nonsense-mutated vectors can be effectively expressed in COS-7 cells. It is concluded that the nonsense-mutated eukaryotic expression vectors of fIX gene have been successfully constructed and can express in COS-7 cells, which provides the material basis for further researches on mechanism and treatment of FIX deficiency and the function defects caused by nonsense mutation.

Rab proteins belong to a subfamily of small GTP-binding proteins of the Ras superfamily, which play an important role in intracellular vesicular traffic. In this study, a rab gene was obtained from Euplotes octocarinatus by polymerase chain reaction (PCR) and RT-PCR. The rab gene from macronucleic DNA was 884 bp in length, including non-coding regions and telomeric sequences at both ends. The rab gene from micronuclear DNA (723 bp), lacking of internal eliminated sequences, was identical to rab gene from macronuclear DNA. RT-PCR showed that the opening reading frame of the rab gene was 663 bp long. The rab gene from macronuclear DNA contained an intron of 60 bp at the position from 153 bp to 212 bp of macronuclear DNA. The rab gene had two in-frame TGAs encoding for cysteine in Euplotes octocarinatus. The rab gene used TAG as stop codon, which was the first report in Euplotes octocarinatus. The result of BLAST in NCBI demonstrates that the Rab shares a homology of 49-52% at the amino acid level with Rab1 proteins from a number of other eukaryote, which suggesting that the Rab is a Rab1 homolog. The rab gene was therefore designated Eo-rab-1N (GenBank accession number: DQ105562). The evolution of Eo-rab-1N was analyzed using phylogenetic tree of amino acids sequences of Rab1 obtained from GenBank.

Translation termination on the ribosome is an essential process for cell viability. This process is maintained by two classes of peptide release factors (RF1/RF2, RF3 and eRF1, eRF3 in prokaryotes and eukaryotes, respectively). In protozoa ciliates Euplotes octocarinatus, an unicellular eukaryotes, universal stop codon UGA is reassigned for cysteine suggesting the specificity of evolution of translation termination system. We cloned two classes of release factors from Euplotes octocarinatus previously. In this paper, three in-frame stop codons UGA in Eo-eRF3 gene were mutated mediated by PCR site directed mutagenesis method. The interaction between eRF1 and eRF3 from E. octocarinatus was assayed in vivo using Yeast Two-hybrid System, which has an advantage of highly sensitivity. The results showed that the eRF1 x eRF3 complex was formed in living cells to function in the process of translation termination, differing from that in prokaryotes in which RF1/RF2 and RF3 function separately. The evolution of translation termination of life-form was analyzed using phylogenetic tree of amino acids sequences of RFs (32 (e) RF1s and 24 (e) RF3s) obtained from GenBank. Two classes of RFs are useful information in analysis of evolution of life-form and further elucidation of mechanism of translation termination of protein synthesis on ribosome.

The expansion of trinucleotide repeats in genome is related to the phthogenesis of several neurodegenerative deseases. A GARP (glutamic acid-rich protein) gene was isolated from the macronuclear plasmid mini library of Euplotes octocarinatus. A micronuclear version of the GARP gene was amplified by polymerase chain reaction. The macronuclear DNA molecule carrying the GARP gene is 460 bp long and shows the characteristics of macronuclear chromosomes of hy- potrichous ciliates. One of the three cysteines is encoded by the opal codon TGA(88-90). The predicted open reading frame encodes a 112-amino acid polypeptide, with a predicted molecular mass of 13 kDa and an isoelectric point of 3.82. Micro- nuclear version of the GARP gene contains two internal eliminated sequences (IES), IES1 and IES2. IES1 is 41 bp long and is flanked by 5'-GA-3' direct repeats. IES2 is 41 bp long and flanked by 5'-TA-3' direct repeats. Transcriptional activity of GARP gene was confirmed by reverse transcription polymerase chain reaction (RT-PCR).