Abstract:Pseudogenes, which have long been described as “fossils”, play a very important role in eukaryotic genomes. Recently, studies on the so called “junk gene” have attracted more attention. Far from being silent, pseudogenes participate in various biological activities, including being a part in the transcription process, or participating in the formation of small interfering RNA (siRNA) which regulated gene expression by means of the RNA-interference pathway. Recent studies have also shown that pseudogenes regulate tumor suppression through competing for the microRNA (miRNA) with their parent genes. However, a deeper understanding of function analysis of pseudogenes depends on the comprehensive and accurate identification. With the sequencing completion of many genomes and the innovation of bioinformatics tools, efficient and precise identification of pseudogenes have become available in a genome-wide scale. Our review focused particularly on the method of pseudogene identification, the mechanism of its regulatory roles and its potential to be applied in directed evolution. Besides, the promising research direction of pseudogenes was proposed.

Abstract:Gene therapy has been considered as a promising method for treatment of many diseases, such as acquired and genetic diseases. At present, there are two major vehicles for gene delivery including viral vectors and nonviral vectors. Viral vectors appear as high gene transfection efficiency, but some deficiencies such as inflammatory responses, recombination and mutagenesis have limited their use. On account of low pathogenicity, safety and cost-effectiveness, nonviral vectors have been attracted much attention. Cationic polymers are one of the nonviral vectors which have been widely studied. This review focuses on the structure of the cationic polymers and the interaction mechanism between the vector and DNA. We try to provide a framework for the future design and synthesis of nonviral vectors with high transfection efficiency and low toxicity for gene therapy.

Abstract:microRNAs (miRNAs) are a family of important small non-coding RNA molecules, which participate in the post transcriptional gene regulation. In this review, the numbers and chromosomal distribution of chicken miRNAs, and the regulation and function of chicken miRNAs in immune, embryo development and virus infection were reviewed. Additionally, the applications of chicken miRNAs were also discussed briefly. We hope it can provide references for further study and use of miRNAs in poultry husbandry fields.

Abstract:Bats are important reservoir animals and more than 60 viruses have been identified in bats with many of them highly pathogenic to human. In order to understand the natural background, genetic diversity of bat viruses in China and discover potential viral pathogens, Solexa sequencing based viral metagenomics focusing on bats tissues was established and to analyze the virome of bats collected from Jilin, Yunnan and Hunan province. By Solexa sequencing, 116 442 324 useful reads were obtained and assembled into 4 872 contigs, of which 8.2% (4 002/4 4872) were annotated to 36 viral families, including 19 vertebrate virus families, 6 plant virus families, 4 insect virus families and 4 phages. Further contigs analyses showed that some adenovirus, bocavirus, picobirnavirus, parvovirus contigs sequences were similar with known viruses. However, part of them shared limited identities to these viruses implying the discovery of new viruses. Moreover, PCR validation of adenovirus and bocavirus confirmed the results obtained by viral metagenomics. This study aimed to understand bat virome in China by viral metagenomics and could be helpful to establish effective surveillance on wildlife-associate zoonoses.

Abstract:We purified a novel mannose binding lectin form Musca domestica pupae by affinity chromatography on Con A-Sepharose 4B and DEAE weak anion-exchange chromatography. By SDS-PAGE, MBL-1 yielded a single band with the molecular weight of 24 kDa. It was a glycoprotein detected by periodic acid-schiff’s staining reaction, with 97.36% protein and 2.1% oligosaccharide. Meanwhile, the results of β-elimination reaction, infrared spectroscopy, atomic force microscopy and protein sequencing instrument show that MBL-1 was an ellipsoidal-shaped monomer with 60?100 nm in diameter. N-glycoside bond linked oligosaccharide chain and the N-terminal blocked peptide chain. Further study suggested that MBL-1 promote the proliferation of macrophage in a concentration-dependent manner. The scanning electron microscope analysis shows that MBL-1 promoted the activation of macrophages. These results show that MBL-1 purified from Musca domestica pupae possesses immune regulation effect, serving a reference basis to develop natural immune-modulator.

Abstract:Breeding of robust industrial Saccharomyces cerevisiae strains with high ethanol tolerance is of great significance for efficient fuel ethanol production. Zinc finger proteins play important roles in gene transcription and translation, and exerting control on the regulation of multiple genes. The sequence and localization of the zinc finger motif can be designed and engineered, and the artificial zinc finger protein can be used to regulate celluar metabolism. Stress tolerance of microbial strains is related to multiple genes. Therefore, it is possible to use artificially-designed zinc finger proteins to breed stress tolerant strains. In this study, a library containing artificial zinc finger protein encoding genes was transformed into the model yeast strain S288c. A recombinant strain named M01 with improved ethanol tolerance was obtained. The plasmid in M01 was isolated, and then transformed into the industrial yeast strain Sc4126. Ethanol tolerance of the recombinant strain of Sc4126 were significantly improved. When high gravity ethanol fermentation using 250 g/L glucose was performed, comparing with the wild-type strain, fermentation time of the recombinant strain was decreased by 24 h and the final ethanol concentration was enhanced by 6.3%. The results of this study demonstrate that artificial zinc finger proteins are able to exert control on stress tolerance of yeast strains, and these results provide basis to construct robust industrial yeast strains for efficient ethanol fermentation.

Abstract:Bacillus sp. TSH1 is a butanol-producing microorganism newly isolated in our laboratory; it can grow and ferment under facultative anaerobic conditions, while sharing similar fermentation pathways and products with Clostridium acetobutylicum. To illustrate the relationships between the products and the enzyme activities in Bacillus sp. TSH1, key butanol- and ethanol-forming enzymes were studied, including butyraldehyde dehydrogenase, butanol dehydrogenase and alcohol dehydrogenase. The activities of the three enzymes increased rapidly after the initiation of fermentation. Activities of three enzymes peaked before 21 h, and simultaneously, product concentrations also began to increase gradually. The maximum activity of alcohol dehydrogenase was 0.054 U/mg at 12 h, butyraldehyde dehydrogenase 0.035 U/mg at 21 h and butanol dehydrogenase 0.055 U/mg at 15 h. The enzyme activities then decreased, but remained constant at a low level after 24 h, while the concentrations of butanol, acetone, and ethanol continued increasing until the end of the fermentation. The results will attribute to the understanding of the butanol metabolic mechanism, and provide a reference for further study of a facultative Bacillus metabolic pathway.

Abstract:Palmitoleic acid (16:1Δ9), an unusual monounsaturated fatty acid, is highly valued for human nutrition, medication and industry. Plant oils containing large amounts of palmitoleic acid are the ideal resource for biodiesel production. To increase accumulation of palmitoleic acid in plant tissues, we used a yeast (Saccharomyees cerevisiae) acyl-CoA-Δ9 desaturase (ScΔ9D) for cytosol- and plastid-targeting expression in tobacco (Nicotiana tabacum L.). By doing this, we also studied the effects of the subcellular-targeted expression of this enzyme on lipid synthesis and metabolism in plant system. Compared to the wild type and vector control plants, the contents of monounsaturated palmitoleic (16:1Δ9) and cis-vaccenic (18:1Δ11) were significantly enhanced in the ScΔ9D-transgenic leaves whereas the levels of saturated palmitic acid (16:0) and polyunsaturated linoleic (18:2) and linolenic (18:3) acids were reduced in the transgenics. Notably, the contents of 16:1Δ9 and 18:1Δ11 in the ScΔ9D plastidal-expressed leaves were 2.7 and 1.9 folds of that in the cytosolic-expressed tissues. Statistical analysis appeared a negative correlation coefficient between 16:0 and 16:1Δ9 levels. Our data indicate that yeast cytosolic acyl-CoA-Δ9 desaturase can convert palmitic (16:0) into palmitoleic acid (16:1Δ9) in high plant cells. Moreover, this effect of the enzyme is stronger with the plastid-targeted expression than the cytosol-target expression. The present study developed a new strategy for high accumulation of ω-7 fatty acids (16:1Δ9 and18:1Δ11) in plant tissues by protein engineering of acyl-CoA-Δ9 desaturase. The findings would particularly benefit the metabolic assembly of the lipid biosynthesis pathway in the large-biomass vegetative organs such as tobacco leaves for the production of high-quality biodiesel.

Abstract:Cytoplasmic male sterility is an important way to utilize wheat heterosis. The purpose of thisstudy was to identify cytoplasmic type of three wheat male sterile lines. Amplified fragment length polymorphism (AFLP) marker technique was used to analyze the wheat mitochondrial DNA. We isolated mitochondria by differential centrifugation and density gradient ultracentrifugation. The results show that the extracted mitochondrial DNA was pure. It was suitable for PCR and genetic analysis. We got 4 pairs of specific primers from 64 primers combinations. Primer E1/M7 amplified 3 specific fragments in ms(Kots)-90-110. Primer E4/M2 generated 2 specific fragments in ms(Ven)-90-110. Primer E7/M6 amplified 2 specific fragments in ms(S)-90-110. Primer E6/M4 produced 2 specific fragments in ms(Kots)-90-110. Four specific primers could be used to identify three cytoplasmic types of Aegilops kotschyi, Ae. ventricosa and Triticum spelta. It provided the molecular basis to further study the mechanism of wheat cytoplasmic male sterility.

Abstract:CD96 (Tactile) is an adhesion receptor expressed mainly on activated T cells, NK cells. As a family member of the immunoglobulin-like cell receptor, CD96 consists of three immunoglobulin-like domains (V1, V2/C and C) in the extracellular region. Recent studies have shown that the 1st IgV domain of CD96 (CD96V1) plays an essential role in cell adhesion and NK cell-mediated killing. In this study, the 1st IgV domain of human CD96 (hCD96V1) was cloned and expressed in Escherichia coli (BL21). The soluble protein was obtained by refolding of the hCD96V1 inclusion bodies. From analytical ultracentrifugation, we could predict that CD96 V1 maily exists as dimer with approximate molecular weight of 26.9?kDa. The protein was then successfully crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.9?? resolution and belonged to space group P21, with unit-cell parameters a=35.1, b=69.5, c= 49.6?, α=γ=90°, β=105.4°.

Abstract:A15, a DNA aptamer with binding specificity for U87 glioma cells stably overexpressing the epidermal growth factor receptor variant III (U87-EGFRvIII), was generated by cell systematic evolution of ligands by exponential enrichment (cell-SELEX) using a random nucleotide library. Subsequently, we established a cell enzyme-linked assay (cell-ELA) to detect the affinity of A15 compared to an EGFR antibody. We used A15 as a detection probe and cultured U87-EGFRvIII cells as targets. Our data indicate that the equilibrium dissociation constants (Kd) for A15 were below 100?nmol/L and had similar affinity compared to an EGFR antibody for U87-EGFRvIII. We demonstrated that the cell-ELA was a useful method to determine the equilibrium dissociation constants (Kd) of aptamers generated by cell-SELEX.

Abstract:Listeria monocytogenes is a pathogenic bacterium, therefore, it is essential for food safety monitoring to establish a rapid and specific detecting method. In this study, immunomagnetic beads and selective medium were combined to detect Listeria monocytogenes at different concentrations (101?105 CFU/mL). Other three types of Listeria spp., Staphylococcus aureus and Vibrio parahaemolyticus were also detected to conduct the cross-reaction analysis. Meanwhile, contaminated milk samples were prepared to explore the limit of detection of immunomagnetic beads combining with selective medium. Results showed that Listeria monocytogenes with the concentration of 103 CFU/mL and above was successfully detected. Milk samples were detected within 6 hours, with a detection limit of 0.7 CFU/mL. The method developed is capable of detecting milk samples within 30 h, which is 38 h faster compared with national standard method with the same sensitivity.

Abstract:To develop a specific, rapid and convenient method based on molecular motor biosensor to detect food-borne rotavirus. A specific probe was encompassed the conservative region of rotavirus’s VP7 segment, and a molecular motor detect device was constructed by connecting probes to F0F1-ATPase molecular motor through biotin-streptavidin system. This biosensor’s sensitivity was 0.005 ng/mL for rotavirus RNA. Extracted virus RNA was conjugated with the biosensor separately, at the same time ATP was synthesized. By comparing fluorescence intensity, we can detect rotavirus RNA in samples. This method possessed specificity for rotavirus, without any cross-reaction with Hepatitis A virus and noroviris, and it could be accomplished within 1 h. We detected 15 samples using this method and the results were compared with RT-PCR results. This method is sensitive and specific for rotavirus, and it can be used to detect food-borne rotavirus.

Abstract:The CBD gene from Trichoderma reesei was cloned into the Corynebacterium glutamicum secretion expression vector pXMJ19-sp, in which green fluorescent protein was inserted to obtain pXMJ19-sp-GFP-CBD. After induced by 0.5 mmol/L IPTG, GFP-CBD was expressed in Corynebacterium glutamicum at high level of 200 mg/L. The GFP-CBD could be purified to high purity with cellulose column. The results indicated CBD can be successfully used in Corynebacterium glutamicum expression system and thus offer an extremely simple, effective and scalable way for production of recombinant proteins.