The myeloid differentiation factor 88 (MyD88) signaling pathway with a variety of regulatory functions, plays a critical role in the occurrence and development of inflammation and tumor immune response. The purpose is to construct a shRNA interference vector of MyD88 gene for porcine, and verify the effect of interference at the level of transcription and protein expression, in order to choose a optimal interference vector. Four pairs of shRNA oligonucleotide sequences were designed and synthesized according to MyD88 gene sequence of pig in GenBank (Accession: KC766424.1) and then were annealed into double-strand and inserted into the plasmid of pYr-1.1 respectively to construct expression vectors, pYr-1.1-pigMyD88-sh1, pYr-1.1-pigMyD88-sh2, pYr-1.1-pigMyD88-sh3, pYr-1.1 -pigMyD88-sh4. After DNA sequencing, four shRNA expression vectors were transfected into pig alveolar macrophage cells. The interference effect of these vectors was confirmed by detecting the expression of MyD88 with Western blot, Real-time PCR and the TNF-α gene expression in MyD88-slienced macrophages after LPS stimulation. The results show that MyD88 shRNA vectors were successfully constructed, and the specific shRNA of porcine MyD88 gene could significantly depressed the expression of porcine MyD88 at the level of mRNA and protein (P<0.05), with interference effects respectively reaching 36%, 67%, 60%, 69%. In contrast to the control group, the gene expression of TNF-α was decreased significantly in MyD88-slienced macrophages after LPS stimulation. These results indicated that the interference effect of MyD88 shRNA vectors was great. These data will provide insight in the role of MyD88, and the basic information to explore the mechanism of immune response related diseases in pigs.
Objective:After construction of the knock-out mutant of MocR family transcription regulator SSU0562 gene in Streptococcus suis serotype 2, the virulence and biological characteristics of the mutant strain was analysised. Methods:Based on the principle of homologous recombination,recombinant gene knock-out vector was constructed consisting of Spcr cassette with the flanking homology regions to the target gene and the isogenic SSU0562-difficient mutant was screened by allelic replacement. The basic biological characteristics between the mutant Δ0562 and the wild type strain 05ZYH33, and the virulence in murine model were compared. Results:PCR analysis and gene sequencing results confirmed the SSU0562 gene was completely replaced by Spcr cassette, showing the gene knock-out mutant strain Δ0562 was successfully constructed. Reverse transcription PCR (RT-PCR) confirmed the absence of SSU0562 in the Δ0562 strain at the transcriptional level. Study of biological characteristics and the virulence showed that there were no significant differences between Δ0562 and 05ZYH33 in terms of hemolytic activity, growth kinetics and pathogenicity. The chain ability of Δ0562 was significantly weaker than 05ZYH33. Conclusion: SSU0562 gene knock-out did not change the virulence of Streptococcus suis serotype 2 05ZYH33, showing that SSU0562 is not the virulence decision factor of Streptococcus suis. The SSU0562 gene was probably involved in the regulation of the chain ability in Streptococcus suis.
Plant tissue-specific promoters, as important regulatory elements, have promising application for bioreactor and crop breeding by genetic modification. Combined barley microarray data and gene digital differential display technology, a root-specific aquaporin gene HvTIP2;1 was obtained after real-time RT-PCR analysis for gene expression in different tissues and under ABA treatment and various abiotic stresses. Meanwhile, isolation and functional analysis of the HvTIP2;1 promoter was conducted. The results showed that HvTIP2;1 was a root-specific gene regulated by plant growth and development, with significantly higher expression at adult stage than at seedling stage. ABA treatment caused up-regulated expression of HvTIP2;1 which decreased after 10h treatment. Under aluminum and manganese metal ion stresses, HvTIP2;1 expressed much higher than the control, with an up-down-up expression pattern. However, it was consistently down-regulated by salt stress. Drought induced HvTIP2;1 expression increase and then decrease dramatically after 24h treatment. A promoter named Hv1310p, upstream of HvTIP2;1 coding domains, was cloned, where some cis-elements related to root-specific expression and plant response to various abiotic stresses were detected using biological software. Using 5' end deletion, two promoter fragments (514bp and 1 258bp) were fused with GUS gene and transformed into tobacco, respectively. Histochemistry staining and quantitative analysis of GUS activity showed that both can drive GUS gene to express specifically in roots.
Illumina Solexa Hi-seq 2000 high-throughput sequencing technology was used to get the comprehensive transcriptome from flower buds of Xanthoceras sorbifolia. A total of 58 311 unigenes were generated, with average length and N50 of 686bp and 1 180bp respectively. Using Blastx against the public databases of Nr and Swiss-Prot, 37 047 unigenes were annotated. 21 264 unigenes were not found in any databases. Unigenes were classified into 25 classes through COG databases. Through GO classification and KEGG pathway enrichment analysis,all of these differentially expressed unigenes were classified into 55 GO terms and 128 metabolism pathways, respectively. In addition, 12 213 potential SSR loci were detected from 9 794 unigene. Of them, occurrence frequency of mono-nucleotide repeats was highest (34.95%), dinucleotide repeat and trinucleotide repeats accounted for 32.74% and 28.64%, respectively. Some unigenes identified from transcriptome data were referred to four major plant flowering regulation pathways including photoperiod pathway, verbalization pathway, GA-dependent pathway and autonomous pathway. to some extent the molecular mechanisms regulating mode with flower bud morphological differentiation of Xanthoceras sorbifolia has been resolved.
The object was to analyze the effects of different combinations of additional carbon and nitrogen resources on the growth and lipid accumulation of Chlorococcum sp. under modified BG-11 culture pretreated by different concentrations of sodium acetate and sodium nitrate. The biomass of Chlorococcum sp. was determined by dry weight method. The features of lipid accumulation were analyzed by lipid productivity, carotenoid productivity and fatty acid methyl ester (FAME) composition. The results showed that Chlorococcum sp. cultivated in the modified BG11 with mass concentrations of sodium acetate 2g/L and sodium nitrate 1g/L had significantly improved algal growth and lipid accumulation. The final biomass, carotenoid and lipid productivity reached 276.19mg/(L·d), 1.347mg/(L·d) and 108.59mg/(L·d), respectively after 16 days' cultivation. It is worth to note that the yield of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) reached 1.58% of total fatty acids.
To improve the yield of ethanol by Thermoanaerobacterium calidifontis Rx1, constructed a engineered mutant Δack. First a recombinant plasmid containing mutation cassettes of pta::ack, was rebuild, and the vector was transformed to cell to disrupt the target genes on the chromosomal via the homologous recombination. Then glucose fermentation, cellobiose fermentation, xylose fermentation, acid hydrolyzate of corncob of Δack mutant and the wild strain were performed respectively to produce ethanol and lactate. The results indicate that the acetate of Δack mutant is much lower as compared with the wild. Dray cell weight of the mutant is always lower than that of the wild under four conditions. However, the yield of ethanol or lactate is more than the wild. When Δack mutant used cellobiose to produce ethanol, the yield is 3.60g/L higher than another three substrates. At the same time, it could be exist approximative 40mmol/L acetate in the hydrolysate, so the output of lactate and ethanol of the wild are more than that with xylose fermentation.
Listeria monocytogenes, an important Gram-positive foodborne pathogen, can form biofilm in the inert or living surface, leading to an enhanced resistance against harsh conditions and a big challenge to be eliminated from food processing facilities. SigB (σB), which is the main stress response factor of Gram-positive bacteria, plays a crucial role in the formation of biofilm. RsbU, a signal (energy and physical chemical signals) transducer protein of the SigB operon in L.monocytogenes, might participate in biofilm formation in L.monocytogenes. To test this hypothesis, the single and (or) double deletion mutants of rsbU and sigB genes were constructed to compare the biofilm formation abilities with the wild-type strain under different temperature (25℃ and 37℃) and nutrition (nutrient-rich BHI medium and nutrient-poor MEM medium).The data show that deletion of RsbU or SigB significantly decreased biofilm formation ability of L.monocytogenes under different temperatures and media. Moreove,low temperature (25℃) and poor nutritional (MEM medium) conditions facilitated RsbU transducing stress signal to active SigB, which consequently act on biofilm formation in L.monocytogenes.
Scaffold was produced by using 3D printer, its functionality of supporting the differentiation of hMSC and in vivo safety was tested. Method: 3D PLGA/HA composite scaffold was printed with 3D printer, the mechanical strength of the 3D scaffold was tested according to the GB/T1040 and GB/T 9341.Its ability of supporting hMSC multiplication and differentiation was validated in vitro, finally the material was assessed of its in vivo safety based on the GB/T 16886.Result: Porous PLGA/HA 3D scaffold was successful made, whose tensile-strength and bending strength is 38MPa and 42MPa, which is about 5.35 and 5.25 times more than that of human cartilage. The 3D material supports the Chondrogenic differentiation of hMSC. Biological safety experiment results proved that the 3D material accord with the standard of medical devices.
In recent years, roller bottle was still used to produce porcine parvovirus. However, it was inefficient, labor-intensive and covered large areas. Now based on the optimization of time of infection (TOI), multiplicity of infection (MOI) and harvest time, porcine parvovirus static production process with PK-15 cells was well developed, that the maximum virus titre could reach 107.5 TCID50/ml. More specifically, by optimizing TOI, the process was successfully transferred from static culture to microcarrier suspension culture, and 5L bioreactor verification experiment was conducted, which enable the maximum virus titer reached 107.2 TCID50/ml. It was the first time to verify that the yield coefficients of lactate to glucose had a positive correlation with virus titre, that it could reach the peak value as soon as the porcine parvovirus at the maximum virus titre. It can be used as an important parameter for virus harvest.
Sox system of Thiobacillus denitrificans plays a vital role in the metabolism of sulfur compounds, SoxYZ coding by the sulfur oxidizing gene cluster (sox) is known to be a sulfur covalently binding protein, which binds sulfur compounds to the other enzymes. The structure of SoxYZ heterodimer, the carrier of sulfur compounds, is constructed by using homology modeling and is proved to be reliable. Analysis of protein interactions find that the Solvent Accessible Surface(SAS) of SoxYZ is 10 922.9Å2, hydrophobicity is 50.85%; the interface between subunits SoxY and SoxZ contains a total of 12 hydrogen bonds and a pi bond which maintain the stability of the three-dimensional structure; the electrostatic potential of SoxYZ surface is obviously complementary, the VDW interaction energy and electrostatic interaction energy of residues at the interface is －80.925 13kcal/mol and －323.856 57kcal/mol, respectively which showed that the electrostatic interaction energy was the main driving force to form the heterodimer and the residues Thr28, Arg31, Lys32, Ser64, Gly65, Val66, Ser67 of SoxZ played an important role in the stability of active site of SoxY.
To overcome the difficulty in genetic transformation of undomesticated intact strains of Bacillus licheniformis, an efficient method combining the use of protoplasts and electroporation was developed. Operational conditions concerning cell growth phase, incubation time with lysozyme, voltage, osmoprotectant, etc., were optimized and applied for the transformation of two undomesticated strains, i.e. B. licheniformis CICC 10181 and B. licheniformis CICC 20204, with both replicative and integrative plasmids. When the late logarithmic-phase culture were digested with lysozyme at a final concentration of 10mg/ml for 40min at 37℃, then pulsed at the voltage of 0.6kV/mm and regenerated with modified DM3 medium containing 0.5mol/L sorbitol or mannitol as osmotic pressure stabilizer, transformation efficiency of B. licheniformis CICC 10181with replicative plasmid pGJ103 and integrative plasmids pAX01 reached 1.1×102CFU/μg DNA and 0.52×102CFU/μg DNA, respectively. In addition, transformation efficiencies of 0.88×102CFU/μg pGJ103 and 0.45×102CFU/μg pAX01 were obtained in another test strain, B. licheniformis CICC 20204. This method provides a valuable and effective tool for molecular genetic modification of interesting wild B. licheniformis strains.
The mammalian oocytes were in vitro fertilization (IVF), the preimplantation embryos (PE) were in vitro cultured (IVC) that was one of important technical in the modern bio-science. The normally to be used medium, one had been called chemically defined medium (CDM). It had been used in a wildly field, frequently. It included in the animal and human's artificial assisted reproductive technical (ART), to research the embryo of earlier developing stage, the transgenic animal, and the animals clone, etc. The review was included in the development history and its constituents of the CDM. There was also included some modified embryo media, and focused on the especial media, which was to be used for culturing the mouse or rat embryo. Meanwhile, the article had introduced the prospects and research direction of CDM, briefly.
FGF9(fibroblast growth factor 9,FGF9) is one of the fibroblast growth factor (FGF) family members.It is widely distributed in various tissues of human body and involved in bone development,angiogenesis, embryonic development,damage repair,cell apoptosis,nerve regeneration,tumor growth and other physiological and pathological processes,and can effectively promote mitosis and cell growth.Current evidence suggests that FGF9 may play a prominent role not only in bone development and growth of cartilage into bone formation and generation,but also has a significant impact on aspects of ovarian cancer,nerve regeneation,gonadal differentiation, there remains much to be discovered and investigated on the functions and mechanisms of FGF18.The research progress in the mechanism of the expression of FGF9 was reviewed and its indication aspects,providing the reference for the further study of FGF9 and its rational development and utilization.
Lipopeptide biosurfactants are small compounds synthesized by various microorganisms as secondary metabolites. The common molecules of such biosurfactants contain both fatty acid chains as the hydrophobic portion and cyclic peptides as the hydrophilic moieties. Due to the amphipathic structures, lipopeptides present promising properties like surface activity and anti-microbial activities and therefore can be applied in chemical, agricultural, pharmaceutical and food industries. Lipopeptide biosurfactants produced by the most studied Bacillus are mainly divided into three families according to the structure of cyclic peptides: surfactin, fengycin and iturin. Surfactins presents outstanding surface activity by significantly reducing the surface tension of water while fengycins and iturins show strong anti-fungal antibiotic activity. Although less known as other lipopeptide biosurfactants, fengycins are well recognized as potential bio-fungicides based on current researches. In this review, the basic structure, synthesis mechanism and fungitoxic activity of fengycins were introduced. Moreover, strategies and current progress on biosynthesis reinforcement were also discussed for further studies.
Séralini's "GM corn carcinogenic study" paper has attracted many concerns and aroused a great controversy in academia and the international community. After recalling the reaction from various communities and analyzing the relevant published literatures, Séralini's research materials, experimental design, data statistics were investigated. The results showed that the materials cannot ensure the uniqueness of its conclusions, and test samples cannot guarantee its conclusions of reproducibility, there can be various interpretations of the results. The results also argued the review system of the journal which hastily published Séralini's paper. It is one of reasons why Séralini's paper still affects international social controversy over genetically modified crops' safety. Furthermore, in which it was cited in the web of science literature, all the study results supported by the experimental data showed that genetically modified crops feeding animals did not significantly affect the health of the animals .Therefore,"GMO's carcinogenic" is a non-scientific conclusion, It is necessary to discuss and resolve controversial issues for scholars and the general public by scientific rational ways.
