Excavating and Expressing the Individuality Culture in the Environmental Landscape Design of Residential Plot——Taking the Environmental Landscape Section of ‘Miaojiang·Yujinyuan’ Residential Plot in Taijiang County of Guizhou Province as an Example

Local culture resources were excavated and expressed in environmental landscape section of ‘Miaojiang ·Yujinyuan’ residential plot in Taijiang County of Guizhou Province, with lusheng, Bronze drum and national architecture as design elements. Making environmental landscape of residential plot, where the minority nationality lived in, possess culture agreement and emotion arrangement.

Establishment of BHK-21 Stable Cell Lines Expressing T7 RNAP and GFP

[Objective] The aim was to establish the BHK-21 stable cell lines expressing T7 RNAP and GFP.[Method]T7 RNAP gene was amplified from E.coli BL21（DE3）and inserted into FG12 vector.The lenti-virus recombinant plasmid FG12-T7 RNAP plasmid was obtained via identification with double enzymes digestion and gene sequencing.The transient expressed T7 RNAP protein was determined by WB in 293T cells transfected with FG12-T7 RNAP plasmid.The recombinant FG12-RNAP lenti-virus was packaged up by transfecting the 293 cells with the recombinant vector FG12-RNAP and the helper plasmids via lipofectamine-2000,which was then used to infect BHK-21 cells.The positive cell clones were obtained after continuous screening by GFP.The expression of T7 RNAP gene was confirmed by Western blot.[Result]The cell line stably expressing the T7 RNAP gene was established by Western blot.[Conclusion]T7 RNAP gene could be stably expressed in eukaryotic cells and it provided a good platform to rescue RNA virus in vivo.

The Differences of Expressing Love between Western and Chinese Culture

Cultures are different between western countries and China,and people's ways of expressing their love are also different.Thes edifferences badly influence well communication between lovers and family members.By comparing the different ways of expressing love between western people and Chinese,this paper tries to make readers deeply recognize and make use of the differences of expressing love.

Cloning One CIPK Gene from a Thermo-Sensitive Genic Self-Incompatible Line in Maize Expressing Under Different Temperatures

A calcineurin B-like protein-interacting protein kinases （CIPK） gene was cloned （EU 424058） from a thermo-sensitive genic self-incompatibility （TSGI） line, HE97, in maize, which was selected for suppression subtractive hybridization （SSH） library of the self-incompatible and self-compatible silks of the TSGI line under different temperatures. The full-length cDNA of the candidate CIPK-like gene consisted of 1 918 nucleotides and contained a 1 332-bp open reading frame in maize. Real-time PCR indicated that the candidate CIPK-like gene was highly expressed in the self-incompatible pollen of the TGSI line, and promoted elevated levels of calcium （Ca2＋）. High Ca2＋ concentrations in the pollen strongly inhibit pollen tube formation in silk of the same plant, thus inducing self-incompatibility （SI） under high temperature. These results implied that the CIPKs-like gene would play an important role in the regulation of HE97 characteristics under different temperatures, perhaps acting as a secondary messenger in the expression of SI in the TGSI line in maize.

MiR-21-5p-expressing bone marrow mesenchymal stem cells alleviate myocardial ischemia/reperfusion injury by regulating the circRNA_0031672/miR-21-5p/programmed cell death protein 4 pathway

BACKGROUND For patients with coronary heart disease,reperfusion treatment strategies are often complicated by ischemia/reperfusion(I/R)injury(IRI),leading to serious organ damage and malfunction.The miR-21/programmed cell death protein 4(PDCD4)pathway is involved in the IRI of cardiomyocytes;however,the aberrant miR-21 expression remains unexplained.Therefore,this study aimed to explore whether circRNA_0031672 downregulates miR-21-5p expression during I/R and to dete-rmine whether miR-21-5p-expressing bone marrow mesenchymal stem cells(BMSCs)reduce myocardial IRI.METHODS CircRNA_0031672,miR-21-5p,and PDCD4 expressions were evaluated in the I/R rat model and hypoxia/re-oxy-genation(H/R)-treated H9C2 cells.Their interactions were subsequently investigated using luciferase reporter and RNA pull-down assays.Methyltransferase-like 3,a methyltransferase catalyzing N6-methyladenosine(m6A),was overexpressed in H9C2 cells to determine whether m6A modification influences miR-21-5p targeting PDCD4.BMSCs stably expressing miR-21 were co-cultured with H9C2 cells to investigate the protective effect of BMSCs on H9C2 cells upon H/R.RESULTS I/R downregulated miR-21-5p expression and upregulated circRNA_0031672 and PDCD4 expressions.CircRNA_0031672 knockdown increased miR-21-5p expression,but repressed PDCD4 expression,indicating that circRNA_0031672 com-petitively bound to miR-21-5p and prevented it from targeting PDCD4 mRNA.The m6A modification regulated PDCD4 expres-sion,but had no effect on miR-21-5p targeting PDCD4.The circRNA_0031672/miR-21-5p/PDCD4 axis regulated myocardial cells viability and apoptosis after H/R treatment;co-culture with miR-21-5p-expressing BMSCs restored miR-21-5p abundance in H9C2 cells and further reduced H9C2 cells apoptosis induced by H/R.CONCLUSIONS We identified a novel circRNA_0031672/miR-21-5p/PDCD4 signaling pathway that mediates the apoptosis of cardiomyocytes and successfully alleviates IRI in myocardial cells by co-culture with miR-21-5p-expressing BMSCs,offering novel insights into the IRI pathogenesis in cardiovascular diseases.

Differential gene expression in nontransgenic and transgenic“M.26”apple overexpressing a peach CBF gene during the transition from eco-dormancy to bud break

The CBF signal pathway is responsible for a significant portion of plant responses to low temperature and freezing.Overexpression of CBF genes in model organisms such as Arabidopsis thaliana enhances abiotic stress tolerance but also reduces growth.In addition to these effects,overexpression of the peach(Prunus persica[L.]Batsch)CBF1 gene in transgenic apple(Malus x domestica Borkh.)line T166 also results in early entry into and late exit from dormancy.Although the regulation of dormancy-induction and dormancy-release occur while the CBF regulon is operative in perennial,woody plants,how overexpression of CBF1 affects these dormancy-related changes in gene expression is incompletely understood.The objective of the present study was to characterize global changes in gene expression in peach CBF1-overexpressing and non-transformed apple bark tissues at different states of dormancy via RNA-seq.RNAseq bioinformatics data was confirmed by RT-qPCR on a number of genes.Results indicate that the greatest number of significantly differentially expressed genes(DEGs)occurred in April when dormancy release and bud break normally occur but are delayed in Line T166.Genes involved in storage and inactivation of auxin,GA,and cytokinin were generally upregulated in T166 in April,while those for biosynthesis,uptake or signal transduction were generally downregulated in T166.Genes for cell division and cambial growth were also downregulated in T166 relative to the non-transformed line.These data suggest that overexpression of the peach CBF1 gene impacts growth hormone homeostasis and as a result the activation of growth in the spring,and most likely growth cessation in the fall as well.

Intramammary expression and therapeutic effect of a human lysozyme-expressing vector for treating bovine mastitis

To develop a gene therapy strategy for treating bovine mastitis, a new mammary-specific vector containing human lysozyme (hLYZ) cDNA and kanamycin resistance gene was constructed for intramammary expression and clinical studies. After one time acupuncture or intracisternal infusion of healthy cows with 400 μg of the p215C3LYZ vector, over 2.0 μg/ml of rhLYZ could be detected by enzymatic assay for about 3 weeks in the milk samples. Western blotting showed that rhLYZ secreted into milk samples from the vector-injected cows had molecular weight similar to that of the natural hLYZ in human colostrums. Twenty days after the primary injection, the quarters were re-injected with the same vector by quarter acupuncture and even higher concentrations of rhLYZ could be detected. Indirect competitive ELISA of milk samples showed that the vector injection did not induce detectable humoral immune response against hLYZ. Clinical studies showed that twice acupuncture of quarters with the p215C3LYZ vector had overt therapeutic effect on clinical and subclinical mastitis previously treated with antibiotics, including disappearance of clinical symptoms and relatively high microbiological cure rates. These data provide a solid rationale for using the vector to develop gene therapy for treating bovine mastitis.

UGPase of Astragalus membranaceus : cDNA Cloning, Analyzing and Expressing in Escherichia coli

On the basis of sequences of UGPase from plants, a cDNA encoding the enzyme was isolated from the hairy root of Astragalus membranaceus (Fisch.) Bunge. The cDNA consisted of 1 831 bp and encoded a polypeptide of 471 amino acid residues with a calculated molecular weight of 51.5 kD and a deduced isoelectric point of 6.01. Then the open read frame of the cDNA was ligated into pET28(a) + vector and expressed in E. coli BL21. SDS_PAGE showed that the expressed protein was ca. 40% in the total bacterial protein. Enzyme activity assay demonstrated that the UGPase activity in the transformed bacteria was 0.50-3.27 times higher than that of the control. Northern blotting revealed that ugp was expressed in the leaf, stem, root and hairy root of A. membranaceus , with a higher level in root and hairy root.

Construction of Plasmids Expressing Sars-CoV Encoding Proteins and Their Effects on Transcription of Hfgl2 Prothrombinase

SARS coronavirus （SARS-CoV） is the etiologic agent of severe acute respiratory syndrome. The aim of this study was to construct Sars-CoV membrane （M）, nucleocapsid （N） and spike 2 （$2） gene eukaryotic expression plasmids, and identify their expression in vitro. Gene fragments encoding N protein, M protein and $2 protein of SARS-CoV were amplified by PCR using cDNA obtained from lung samples of SARS patients as template, and subcloned into pcDNA3.1 vector to form eukaryotic expression plasmids. SARS-CoV protein eukaryotic expression plasmids were transfected respectively into CHO cells. Immunohistochemistry was employed to detect the expression of the structural proteins of SARS-CoV in transfected cells. SARS-CoV protein eukaryotic expression plasmids were successfully constructed by identification with digestion of restriction enzymes and sequencing. M, N and S2 proteins of SARS-CoV were detected in the cytoplasm of transfected CHO cells. It was concluded that these recombinant eukaryotic expression plasmids were constructed successfully, and SARS-CoV encoding proteins could activate transcription and expression of hfgl2 gene.

Transcriptional profiling reveals multiple defense responses in downy mildew-resistant transgenic grapevine expressing a TIR-NBS-LRR gene located at the MrRUN1/MrRPV1 locus

Grapevine downy mildew(DM)is a destructive oomycete disease of viticulture worldwide.MrRPV1 is a typical TIR-NBS-LRR type DM disease resistance gene cloned from the wild North American grapevine species Muscadinia rotundifolia.However,the molecular basis of resistance mediated by MrRPV1 remains poorly understood.Downy mildew-susceptible Vitis vinifera cv.Shiraz was transformed with a genomic fragment containing MrRPV1 to produce DM-resistant transgenic Shiraz lines.Comparative transcriptome analysis was used to compare the transcriptome profiles of the resistant and susceptible genotypes after DM infection.Transcriptome modulation during the response to P.viticola infection was more rapid,and more genes were induced in MrRPV1-transgenic Shiraz than in wild-type plants.In DM-infected MrRPV1-transgenic plants,activation of genes associated with Ca^(2+)release and ROS production was the earliest transcriptional response.Functional analysis of differentially expressed genes revealed that key genes related to multiple phytohormone signaling pathways and secondary metabolism were highly induced during infection.Coexpression network and motif enrichment analysis showed that WRKY and MYB transcription factors strongly coexpress with stilbene synthase(VvSTS)genes during defense against P.viticola in MrRPV1-transgenic plants.Taken together,these findings indicate that multiple pathways play important roles in MrRPV1-mediated resistance to downy mildew.

Cloning,Expressing,and Hemolysis of tdh,trh and tlh Genes of Vibrio parahaemolyticus

Vibrio parahaemolyticus (VP) is one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea,Fennerpenaeus chinensis, and shellfish in coastal areas of China. Several types of hemolysins produced by Vp have been characterized as major virulence factors.They are thermostable direct hemolysin (TDH),TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). In this study, we cloned tdh, trh, and tlh genes from the genome DNA of VP by polymerase chain reaction (PCR).We ligated the three genes into prokaryotic expression vector pET-28a (+),and transformed the recombinant plasmids into Es-cherichia coli BL21 (DE3). The expression of recombinant proteins was induced by isopropyl-β-D-thiogalacto-pyranoside (IPTG). The recombinant proteins were expressed in a form of inclusion bodies and thus purified with Ni-NTA affinity chromatography. Western blotting results showed that recombinant proteins,TDH, TRH and TLH, could be recognized by rabbit anti-VP serum. The three purified proteins were renatured by gradient dialysis.The renatured proteins exhibited hemolytic activity except for TLH in the presence of phosphatidylcholine. These results not only are helpful for better understanding these genes' functions under a single factor level, but also provide evidence for VP vaccine engineering.

Tomato expressing Arabidopsis glutaredoxin gene AtGRXS17 confers tolerance to chilling stress via modulating cold responsive components

Chilling stress is a production constraint of tomato,a tropical origin,chilling-sensitive horticultural crop.The development of chilling tolerant tomato thus has significant potential to impact tomato production.Glutaredoxins(GRXs)are ubiquitous oxidoreductases,which utilize the reducing power of glutathione to reduce disulfide bonds of substrate proteins and maintain cellular redox homeostasis.Here,we report that tomato expressing Arabidopsis GRX gene AtGRXS17 conferred tolerance to chilling stress without adverse effects on growth and development.AtGRXS17-expressing tomato plants displayed lower ion leakage,higher maximal photochemical efficiency of photosystem II(Fv/Fm)and increased accumulation of soluble sugar compared with wild-type plants after the chilling stress challenge.Furthermore,chilling tolerance was correlated with increased antioxidant enzyme activities and reduced H2O2 accumulation.At the same time,temporal expression patterns of the endogenous C-repeat/DRE-binding factor 1(SlCBF1)and CBF mediated-cold regulated genes were not altered in AtGRXS17-expressing plants when compared with wild-type plants,and proline concentrations remained unchanged relative to wild-type plants under chilling stress.Green fluorescent protein-AtGRXS17 fusion proteins,which were initially localized in the cytoplasm,migrated into the nucleus during chilling stress,reflecting a possible role of AtGRXS17 in nuclear signaling of chilling stress responses.Together,our findings demonstrate that genetically engineered tomato plants expressing AtGRXS17 can enhance chilling tolerance and suggest a genetic engineering strategy to improve chilling tolerance without yield penalty across different crop species.

Clay nanosheet-mediated delivery of recombinant plasmids expressing artificial miRNAs via leaf spray to prevent infection by plant DNA viruses

Whitefly-transmitted begomoviruses are economically important plant pathogens that cause severe problems in many crop plants,such as tomato,papaya,cotton,and tobacco.Tomato yellow leaf curl virus(TYLCV)is a typical monopartite begomovirus that has been extensively studied,but methods that can efficiently control begomoviruses are still scarce.In this study,we combined artificial microRNA(amiRNA)-mediated silencing technology and clay nanosheetmediated delivery by spraying and developed a method for efficiently preventing TYLCV infection in tomato plants.We designed three amiRNAs that target different regions of TYLCV to silence virus-produced transcripts.Three plant expression vectors expressing pre-amiRNAs were constructed,and recombinant plasmid DNAs(pDNAs)were loaded onto nontoxic and degradable layered double hydroxide(LDH)clay nanosheets.LDH nanosheets containing multiple pDNAs were sprayed onto plant leaves.We found that the designed amiRNAs were significantly accumulated in leaves 7 days after spraying,while the pDNAs were sustainably detected for 35 days after the spray,suggesting that the LDH nanosheets released pDNAs in a sustained manner,protected pDNAs from degradation and efficiently delivered pDNAs into plant cells.Importantly,when the LDH nanosheets coated with pDNAs were sprayed onto plants infected by TYLCV,both the disease severity and TYLCV viral concentration in sprayed plants were significantly decreased during the 35 days,while the levels of H_(2)O_(2) were significantly increased in those plants.Taken together,these results indicate that LDH nanosheets loaded with pDNAs expressing amiRNAs can be a sustainable and promising tool for begomovirus control.

EXPRESSING HUMAN MATURED BRAIN-DERIVED NEUROTROPHIC FACTOR GENE IN E.Coli AND DETERMINING ITS BIOACTIVITY

Objective Expressing the human matured brain-derived neurotrophic factor (mBDNF) gene in E. Coli and determining its bioactivity. Methods The resulting gene of mBDNF was subcloned into the EcoRI-BamHI site or the expression vector plasmid pBV220. The ligation products were used to transform the competent E. Coli DH5a. The proteins or mBDNF were experessed by temperature inducing. The expression products were dealed with solubilizing inclusion bodies and refolding protein. It was introduced into the embryonic chicken DRG to test whether the expressed mBDNF is a biologically active protein. Results The recombinant plasmid pBV/mBDNF was success- fully constructed. By temperature inducing, under the control of the bacteriophage λPL promoter, the experessed mBDNF protein was a 14Kd non-fusion protein,which existed in E. Coli as inclusion bodies. The size or expressed mBDNF is identical to the prediction. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of embryonic 8-day-old chicken DRG neurons as compared to control. Conclusion Tke mBDNF gene can be expressed bioactively in E. Coli.

Enhanced Riboflavin Production by Expressing Heterologous Riboflavin Operon from B.cereus ATCC14579 in Bacillus subtilis

Fragment containing the whole riboflavin(rib)operons of B.cereus ATCC14579 was detected from GenBank and annotated.The rib operon of ATCC14579 was cloned with Pn,its native promoter,or with P43,the vegetative growth promoter,into the plasmid.Expression analysis showed that heterologous rib operon was operative in B.subtilis.Integrative plasmid with P43-rib fragment was integrated into the chromosome of B.subtilis RH33,yielding transformant B.subtilis PY.With optimized medium components,4.3 g·L -1 of riboflavin was achieved in batch culture of B.subtilis PY,which was 27%enhancement compared to the host strain.Real-time reverse transcription polymerase chain reaction(RT-PCR)analysis indicated that the transcriptional level of ribA maintained 2.8-fold higher with the expression of herterologous rib operon.Furthermore,the stability of B.subtilis PY was increased form 45%to 87%.The high transcriptional level of rib gene and higher stability of B.subtilis PY could explain the increased riboflavin production.

Melanopsin expression is an indicator of the well-being of melanopsin-expressing retinal ganglion cells but not of their viability

Light is an electromagnetic stimulus that in mammals is sensed by specialized neurons in the retina.The physiological response to light encompasses two fundamental and different functional outputs：image-forming and non-image forming.

Construction of heat shock protein 90α expressing plasmid and the effect of the protein on tumor growth in vivo

Heat shock protein 90 (Hsp90) is a major heatshock protein whose functions are necessary for livingorganisms, including both procaryotes and eucaryotes.Human Hsp90 consists of two forms, Hsp90α andHsp90β. Expression of Hsp90α appears to be inducible bydifferent stimuli. Adenovirus EIA gene products andtransformation with the H-ras oncogene can induce aselective overexpression of Hsp90α. Hsp90α was

Establishment of an artificial β-cell line expressing insulin under the control of doxycycline

AIM: Artificial beta-cell lines may offer an abundant source of cells for the treatment of type I diabetes, but insulin secretion in beta-cells is tightly regulated in physiological conditions. The Tet-On system is a "gene switch" system, which can induce gene expression by administration of tetracycline (Tet) derivatives such as doxcycline (Dox). Using this system, we established 293 cells to an artificial cell line secreting insulin in response to stimulation by Dox. METHODS: The mutated proinsulin cDNA was obtained from plasmid pcDNA3.1/C-mINS by the polymerase chain reaction (PCR), and was inserted downstream from the promoter on the expression vector pTRE2, to construct a recombined expression vector pTRE2mINS. The promoter on pTRE2 consists of the tetracycline-response element and the CMV minimal promoter and is thus activated by the reverse tetracycline-controlled transactivator (rtTA) when Dox is administrated. pTRE2mINS and plasmid pTK-Hyg encoding hygromycin were co-transfected in the tet293 cells, which express rtTA stably. Following hygromycin screening, the survived cells expressing insulin were selected and enriched. Dox was used to control the expression of insulin in these cells. At the levels of mRNA and protein, the regulating effect of Dox in culture medium on the expression of proinsulin gene was estimated respectively with Northern blot, RT-PCR, and radioimmunoassay. RESULTS: From the 28 hygromycin-resistant cell strains, we selected one cell strain (tet293/Ins6) secreting insulin not only automatically, but in response to stimulation by Dox. The amount on insulin secretion was dependent on the Dox dose (0,10,100,200,400,800 and 1000 microg.L(-1)), the level of insulin secreted by the cells treated with Dox (1000 microg.L(-1)) was 241.0pU.d(-1).cell(-1) , which was 25-fold that of 9.7pU.d(-1).cell(-1) without Dox treatment. Northern blot analyses and RT-PCR further confirmed that the transcription of insulin gene had already been up-regulated after exposing tet293/Ins6 cells to Dox for 15 minutes, and was also induced in a dose-dependent manner. However, the concentration of insulin in the media did not increase significantly until 5 hours following the addition of Dox. CONCLUSION: Human proinsulin gene was transfected successfully and expressed efficiently in 293 cells, and the expression was modulated by tetracycline and its derivatives, improving the accuracy, safety, and reliability of gene therapy, suggesting that conditional establishment of artificial beta-cells may be a useful approach to develop cellular therapy for diabetes mellitus.

Improvements of Fiber Yield and Fiber Fineness by Expressing the iaaM Gene in Cotton Seed Coat

Cotton,the most important natural fiber crop in the world,is a mainstay in China's economy.However,for over two decades,cotton yields both in China and U.S.have been at a plateau.

Methods of Expressing Knowledge in CPDES

Knowledge bank is the core of an expert system. In a coal preparation plant design expert system (CPDES), it is difficult to make up the knowledge bank. There is terrible huge array of knowledge for coal preparation plant design. Some of them can eveu not be listed into distinct entries with common expressing methods. Some others would be tangled and tedious if unsuitable methods were used.The thesis explained how the expert knowledge for coal preparation plant design is accePted by computer, and how the different expression of knowledge is selected according to its characteristics.