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Soil microorganisms are important components of terrestrial ecosystems, affecting soil formation and fertility, plant growth and stress tolerance, nutrient turnover and carbon storage. In this study, we collected soil samples (humus layer, 0-10 cm, 10-20 cm, 20-40 cm, and 40-80 cm) from Caragana jubata shrubland in Shanxi subalpine to explore the composition, diversity, and assembly of soil bacterial communities at different depths across the soil profile. The results showed that Actinomycota (19%-28%), Chloromycota (10%-36%) and Acidobacteria (15%-24%), and Proteobacteria (9%-25%) were the dominant bacterial phyla. α-diversity of soil bacterial community significantly decreased with the increases of soil depth. Soil bacterial β-diversity varied across different soil depths. Soil pH, water content, and enzyme activity were the main ecological factors affecting the distribution of soil bacterial communities. Soil bacterial communities had more complex interactions in humus layer and 0-10 cm layer. On the whole, soil bacterial communities were dominated by coexistence in C. jubata shrubland, and the soil bacterial community assembly was driven by random process.

Microbial communities are the key component to maintaining the structure and function of forest soil ecosystems. The vertical distribution of bacterial communities on the soil profile has an important impact on forest soil carbon pools and soil nutrient cycling. Using Illumina MiSeq high-throughput sequencing technology, we analyzed the characteristics of bacterial communities in the humus layer and 0-80 cm soil layer of Larix principis-rupprechtii in Luya Mountain, China, to explore the driving mechanisms affecting the structure of bacterial communities in soil profiles. The results showed that the α diversity of bacterial communities decreased significantly with increasing soil depth, and community structure differed significantly across soil profiles. The relative abundance of Actinobacteria and Proteobacteria decreased with increased soil depth, whereas the relative abundance of Acidobacteria and Chloroflexi increased with the increase in soil depth. The results of RDA analysis showed that soil NH + 4 , TC, TS, WCS, pH, NO - 3 , and TP were important factors determining the bacterial community structure of the soil profile, among which soil pH had the most significant effect. Molecular ecological network analysis showed that the complexity of bacterial communities in the litter layer and subsurface soil (10-20 cm) was relatively high, whereas the complexity of bacterial communities in deep soil (40-80 cm) was relatively low. Proteobacteria, Acidobacteria, Chloroflexi, and Actinobacteria played important roles in the structure and stability of soil bacterial communities in Larch. The species function prediction of Tax4Fun showed a gradual decline in microbial metabolic capacity along the soil profile. In conclusion, soil bacterial community structure showed a certain distribution pattern along the vertical profile of soil, the community complexity gradually decreased, and the unique bacterial groups of deep soil and surface soil were significantly different.

Although soil microbes play a key role in grassland ecosystem functioning, the response of their diversity to grassland degradation has not been fully investigated. Here, we used shotgun metagenomic sequencing to analyze the characteristics and influencing factors of soil microbial taxonomic and functional diversity at four different degradation stages[i.e., non-degraded (ND), lightly degraded (LD), moderately degraded (MD), and heavily degraded (HD)]of subalpine meadow in the Mount Wutai. The results showed that there were significant differences in the relative abundances of Actinobacteria, Bacteroidetes, Nitrospirae, and Parcubacteria among the four subalpine grasslands with different degradation degrees ( P <0.05).Compared with that in ND, the degraded meadows increased the proportion of genes related to carbon metabolism, biosynthesis of amino acids, pyruvate metabolism, citric acid cycle, propanoate metabolism, butanoate metabolism, and fatty acid metabolism ( P <0.05), indicating that the degradation of subalpine grassland changed the metabolic potential of energy metabolism and the nutrient cycle of the soil microbial community. Grassland degradation changed soil microbial taxonomic and functional α diversity, especially in MD and HD.Grassland degradation resulted in significant changes in the taxonomic and functional compositions of the microbial communities. The total nitrogen, pH, and soil organic carbon significantly affected the taxonomic and functional compositions of the microbial communities.The β diversity of the plant community was significantly correlated with the taxonomic and functional β diversity of the microbial community ( P <0.05), indicating strong coupling. The results of this study revealed the changes and driving mechanisms of subsurface microbial taxonomic and functional diversity during grassland degradation, which can provide a theoretical basis for subalpine meadow protection and ecological restoration.

To reveal the assembly mechanisms of soil protozoan community in subalpine forest ecosystems, we analyzed the composition and diversity of protozoan communities and their drivers at the six strata (the litter profile, humus profile, 0-10 cm, 10-20 cm, 20-40 cm and 40-80 cm) of soil profiles in subalpine Larix principis-rupprechtii forest in Luya Mountain using Illumina Miseq high-throughput sequencing technology. The results showed that protozoa in the soil profiles belonged to 335 genera, 206 families, 114 orders, 57 classes, 21 phyla, and 8 kingdoms. There were five dominant phyla (relative abundance >1%) and 10 dominant families (relative abundance >5%). The α diversity decreased significantly with increasing soil depth. Results of PCoA analysis showed that the spatial composition and structure of protozoan community differed significantly across soil depths. The results of RDA analysis showed that soil pH and soil water content were important factors driving protozoan community structure across soil profile. Null model analysis suggested that the heterogeneous selection dominated the processes of protozoan community assemblage. Molecular ecological network analysis revealed that the complexity of soil proto-zoan communities decreased continuously with increasing depth. These results elucidate the assembly mechanism of soil microbial community in subalpine forest ecosystem.

Planktonic and epiphytic bacterial communities play an important role in wetland nitrogen pollutant removal and water purification, yet their community dynamics are far from understood compared with those of the wetland soil bacterial community. Taking the planktonic bacterial community in the Yuguqiao constructed wetland and the epiphytic bacterial community on the leaf surface of the common submerged plant Vallisneria natans as the research objects, the composition, structure, and functional diversity of planktonic and epiphytic bacterial communities were analyzed using high-throughput sequencing. The results showed that the compositions of the planktonic and epiphytic bacterial communities were significantly different, with more heterotrophic and denitrifying bacteria present in the epiphytic bacterial community than in the planktonic bacterial community. The α diversity of the planktonic bacterial community was significantly different among the three sampling sites but not in the epiphytic bacterial community. In general, the OTU index and Shannon index of the epiphytic bacterial community were significantly higher than those of the planktonic bacterial community, and they had obvious spatial heterogeneity. RDA analysis showed that DO, IC, TP, NH + 4 , and TOC had important effects on the structural changes of both planktonic and epiphytic bacterial communities but had a greater impact on planktonic bacterial communities. Co-occurrence network analysis showed that the epiphytic bacterial community had more niche differentiation, a more stable network, and stronger resistance to external disturbance. The results of FAPROTAX functional prediction analysis showed that the nitrogen cycling, especially denitrification of the epiphytic bacterial community, was significantly greater than that of the planktonic bacterial community. The results of this study revealed the driving mechanism for maintaining the diversity of planktonic and epiphytic bacterial communities, which can provide a scientific basis for excavating and utilizing planktonic and epiphytic bacterial community resources in the construction of constructed wetlands to improve the efficiency of water purification.

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 ( P< 0.05). Redundancy analysis (RDA) showed that soil water content, total nitrogen, plant richness, and ammonium nitrogen were significantly correlated with the compositions and structures of fungal communities ( P< 0.05). There were significant correlations between α diversity and β diversity between plant and fungal communities ( P <0.05), indicating strong coupling. The results of our study provide a theoretical basis for further research on the changes in soil fungal communities and their driving mechanism in different degradation stages of subalpine meadows.

Objective: To investigate the molecular mechanism in stable cell strains expressing Mini-hF9 gene with nonsense mutation., Methods: 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., Results: 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., Conclusion: The nonsense associated altered splicing (NAS) pathway, which generated alternately spliced transcripts, might be triggered in coagulation factor IX gene with nonsense mutation.

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 Bothriochloa ischaemum as the research subject in a copper tailings dam. Using high-throughput sequencing, we investigated the structures of the fungal communities and diversities in the phyllosphere and rhizosphere of B. ischaemum . This study also explored the effects of heavy metal content on fungal community characteristics. The results showed that Ascomycota and Basidiomycota were the dominant phyla in the phyllosphere and rhizosphere of B. ischaemum . The diversities and richness of the rhizosphere fungal community were higher than that of the phyllosphere fungal community. The diversities of rhizosphere and phyllosphere fungal communities was affected by different heavy metals. Phyllosphere fungal diversity was mainly affected by the content of Zn and Cu in leaves, and the content of Pb in roots was the key factor affecting the diversity of the rhizophere fungal community. Furthermore, Pleosporaceae had a very significant positive correlation with Cd in the phyllosphere, and Nectriaceae had a significant positive correlation with Zn in the rhizosphere. These fungal communities could be used as indicators of ecological recovery in areas with heavy metal pollution. The results could provide an ecological basis for the exploration and utilization of phyllosphere or rhizosphere fungi resources during ecological restoration processes. This study also provides guidance for selecting the plant-microbial symbionts during ecological restoration in areas with heavy metal pollution.

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 4 + ) contestation were significantly different among the different sampling depths during different months. Bacterial abundance was the highest in August and the lowest in November, and the abundance was higher in the middle water layer (2 m, 4 m, and 6 m depths), but relatively low in the surface layer and bottom layer (0 m and 8 m depths, respectively). The α diversity index of bacterial communities had significant differences among the different months and depths, and showed an initial decreasing trend and then an increasing trend from April to December. A PERMANOVA test showed that the spatial distribution of bacterial communities was significantly different among depths ( P <0.001). The results of redundancy analysis and variation partitioning indicated that environmental selection and diffusion limitation had an effect on the maintenance of the diversity patterns of bacterial communities at the different depths of GH. However, the relative effect of environmental factors was stronger, of which the concentration of NO 3 - , NO 2 - , and NH 4 + were the main influencing factors. In conclusion, the bacterial communities in GH subalpine lake showed clear spatio-temporal distribution patterns, and environmental variables had a significant effect on shaping the community diversity.

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 4 + -N), total carbon (TC), and inorganic carbon (IC) of GH were significantly higher than in the other two lakes. The fungal community was mainly composed of Ascomycota (0.82%-21.05%), Basidiomycota (1.26%-11.79%), Chytridiomycota (0.42%-4.26%), and Rozellomycota (0.11%-0.33%). Cystobasidiomycetes, Dothideomycetes, Chytridiomycetes, and Sordariomycetes were the dominant classes shared by the three lakes. The α -diversity index and the relative abundance of dominant classes were significantly different among the three lakes ( P <0.05), but there were no significant differences between the various depths on each lake. The results of the ANOSIM analysis showed that the β -diversity of the fungal communities were significantly different ( r =0.99, P <0.01) among the lakes. There was also expressive differences at various depths on MYH ( r =0.98, P <0.01) and GH ( r =0.25, P <0.05), but no significant difference in PPH ( r =0.23, P >0.05). The analysis results of redundancy and variation partitioning showed that the β -diversity pattern of fungal communities in small region areas (among the three lakes) and local areas (different depths of MYH) were driven by environmental selection and dispersal limitation. However, the relative role of environmental selection was more significant, with water pH, dissolved oxygen (DO), TC, and EC being the main influencing factors. The results of the null model analysis showed that the interspecific interactions promoted the maintenance of the β -diversity pattern of the fungal community in GH. In summary, the β -diversity pattern of fungal communities in the subalpine lakes was mainly driven by a deterministic process.

The community assembly process, which purports that the trade-off between the stochastic process and the deterministic process, is the central issue of community ecology, and is one of the most controversial ecological issues. The current research investigated the fungal community in the Larix principis-rupprechtii forests of the Guandi Mountains and elucidated the relative role of the stochastic and deterministic processes in the assembly of soil fungal community on a local scale. The correlation analysis and redundancy analysis of the physicochemical factors in soil and dominant fungal phyla, as well as the structural equation model analysis, showed that these physicochemical factors and aboveground vegetation diversity had significant effects on fungal communities. The direct effect of vegetation diversity on fungal community structure was the most (1.1858). It is inferred that the determination process (environmental selection) has a certain influence on the assembly of fungal communities. The β -diversity of fungal community shows a distance-decay pattern; thus, it can be concluded that the stochastic process (dispersal limitation) has a certain effect on the assembly of fungal communities. Null model analysis confirmed that the deterministic process was the main driving factor for the assembly of the fungal communities in the study area, and their relative importance varied along with altitudinal gradient. The null deviations in the study area were negative, suggesting that habitat filtering was the driving factor of the assembly of fungal communities. Overall, the deterministic versus stochastic processes jointly drive the assembly of fungal communities in the study area, while the deterministic processes triumph.

Mining for metal and mineral resources lead to the rapid rise of tailings dams and caused serious damage to the ecological environment of the mining area. Soil physicochemical characteristics and enzyme activities were important indexes for ecosystem functions, and they were also important factors in evaluating soil restoration qualities. We selected nine sub-dams of the Eighteen River copper tailings in Yuanqu County, and analyzed the relationship between soil physicochemical properties and soil enzyme activities. The results showed that there were great differences in soil physicochemical properties over different reclaimed years, and as the reclaimed years passed, soil nutrient contents significantly increased. There were significant negative correlations between catalase and the ratio of soil carbon and nitrogen, and urease was positively correlated to total nitrogen and soil moisture. Phosphatase and sucrose demonstrated no significant relationships with soil physicochemical factors. Copper content gradually accumulated in soil as the restoration period of sub-dams increased. Arsenic and cadmium content increased initially and then decreased before they gradually reached a stable level. In addition, there was no significant difference in zinc content among different sub-dams. Together, these results provide the ecological basis for further studies in soil ecosystem restoration and degradation mechanisms in copper tailings.

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 3 - -N, NO 2 -N), plant community, and soil enzyme activities jointly drove bacterial community assembly on the copper mine tailings.

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.

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.

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 Larix principis-rupprechtii forest, Picea wilsonii forest, Pinus tabulaeformis forest and Betula spp. forest, respectively, in Pangquangou Nature Reserve in Guandi Mountains with Illumina high-throughput sequencing technology. Meanwhile, soil environmental factors and diversity of undergrowth plants were determined to analyze the relationship between fungi community structure and vegetation as well as soil environmental factors. The results showed that:①There were seven eumycota and thirty-three advantageous fungal genera in the five sample sites; ②Redundancy analysis results showed that soil pH, temperature, moisture, total nitrogen, the content of NH 4 + , total carbon, invertase activity, urease activity, undergrowth dominance and evenness were significantly associated with soil fungi community structure; ③Cluster analysis and principal component analysis showed that forest vegetation type, soil environmental factors and undergrowth had significant effects on soil fungi community structure; ④The results of PCNM analysis showed that at a local scale, dispersal limitation had no significant influence on fungi community structure in the study area. The forest soil fungi community structure in the study area was significantly affected by environmental selection (soil pH, temperature, moisture, total nitrogen, the content of NH 4 + , total carbon, invertase activity, urease activity, undergrowth dominance and evenness, forest type).

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.

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.

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.

In eukaryotes, eRF3 participates translation termination and belongs to the superfamily of GTPase. In this work, dissociation constants for E. octocarinatus eRF3 binding to nucleosides in presence and absence of eRF1a were determined using fluorescence spectra methods. Furthermore, the GTP hydrolyzing assay of Eo-eRF3 was carried out by HPLC methods and the kinetic parameter for GTP hydrolysis by eRF3 was determined. The results showed eRF1a could promote GTP binding to eRF3 and hydrolyzing GTP activity of eRF3. The observation is consistent with the data from human. Whereas E. octocarinatus eRF3 alone can bind GTP in contrast to no GTP binding observed in the absence of eRF1 in human eRF3. The affinity for Eo-eRF3 binding nucleotides is different from that in human. Structure model and amino acids sequence alignment of potential G domains indicated these different may be due to Valine 317 and Glutamate 452 displacing conserved Glycine and Lysine, which were 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.

The expansion of trinucleotide repeats in genome is related to the phthogenesis of several neurodegenerative diseases. 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 hypotrichous 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. Micronuclear 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).

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.