A Preliminary Analysis on the Construction and Expression of Eukaryotic Expression Vectors of Mytilin and Myticin from Mytilus coruscus

[Objective] The aim was to study the construction and expression of eukaryotic expression vectors of antibacterial peptides （mytilin and myticin） from Mytilus coruscus.[Method] By the screening of antibacterial peptide genes of mytilin and myticin of Mytilus coruscus,five antibacterial peptide genes were selected.Then,the relative eukaryotic expressing vectors were constructed by the use of PCR technique and DNA recombinant technology.Subsequently,they were transferred in to S78 Saccharomyces cerevisia by using LiAC transformation method,and then preliminary expressing analysis was carried out.[Result] Five eukaryotic expressing vectors of antibacterial peptides from Mytilus coruscus were successfully constructed,and the results of mRNA detection revealed that the five antibacterial peptides from Mytilus coruscus were successfully transcribed.[Conclusion] The results provide a basis for using genetic engineering to express antibacterial peptides of mytilin and myticin from Mytilus coruscus,and for developing the further study of antibacterial peptides from Mytilus coruscus based on this.

Construction of Prokaryotic Expression Vectors of EBP1 Gene from Nervilia Fordii (Hance) Schltr.

[Objective] To construct prokaryotic expression vectors encoding gene Erb3binding protein （EBP1）, which plays important roles in regulating plant organ size from Nervilia fordii （Hance） Schltr. [Methods] PCR products of NfEBP1 with particular restriction sites and expression vectors, pET-28 and pET-16b were digested. Ligation, transformation and selection were performed to construct the recombinant plasmids pET-28-NfEBP1 and pET-16-NfEBP1. The recombinant plasmids were transformed into E. coli BL21 using heat -shock transformation. [Results] Recombinant plasmids pET-28-NfEBP1-1188 and pET-16-NfEBP1-1188 were constructed and transformed into expressional host cells, E. coli BL21, and validated by colony PCR, sequencing and double digestion. [Conclusion] Prokaryotic expression vectors of EBP1 gene from N. fordii were successfully constructed, which laid the foundation for characterization of the gene function.

Establishment of the Agrobacterium-mediated Genetic Transformation System of Ginkgo biloba and the Construction of the Expression Vector of Gb-DXR

[Objective] The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba mediated by Agrobacterium.[Method] Taking the mature embryos of Ginkgo biloba seeds as explants,after 48 hours＇ pre-cultivation on MS medium in the absence of phytohormone,GUS gene was transmitted into embryos of Ginkgo biloba mediated by three kinds of Agrobacterium.Transient expression of GUS gene activity was observed through histochemical staining,and the influencing factors of the expression of GUS gene were analyzed.And the expression vector of 1-deoxy-D-xylulose-5-phosphate reductoisomerase in the biosynthesis approach of biobalide precursor of Ginkgo biloba was constructed.[Result] A more suitable genetic transformation scheme was obtained as follows：taking embryos of Ginkgo biloba as explants,using EHA105 Agrobacterium with pCAMBIA1304＋ for infection,co-culture for 3 days and GUS staining.The results showed that transient expression rate of GUS after transformation was higher.[Conclusion] The research provide a more effective method for further study on the transgene of Ginkgo biloba.

DNA immune responses induced by codelivery of IL-12 expression vectors with hepatitis C structural antigens

Objective: To demonstrate the utility of DNA vaccines for the tailored methods, the efficacy of enhanced immune responses, and the types of increased im- mune responses. Methods: Four recombinant plasmids constructed in- cluded the coding regions for the core protein (pC) and for the core, E_1 and E_2 together (pCE_1E_2), IL- 12 p35 and p40. These plasmids were transfected into mammalian cells to test their protein expression and were injected into the quadriceps muscles of BALB/ C mice for measurement of specific antibodies and cytotoxic T-lymphocyte (CTL) responses. Results: All the recombinant plasmids were shown to express specific antigens stably in mammalian cells. Codelivery of pIL-12 expression cassettes with pC and pCE_1E_2 in mice resulted in the enhancement of Ag-dependent CTL responses and the reduction of specific Ab response. The CTL activity was: pC= 18.65%±5.71%, pCE_1E_2=20.07%±11.11%, pC +pIL-12=60.11%±17.37%, pCE_1E_2+pIL-12= 67.48%±15.57%, respectively. The average A val- ues of anti-HCV were pC=0.415±0.127, pCE_1E_2= 0.358±0.096, pC+pIL-12=0.210±0.086, pCE_1E_2 +pIL-12=0.258±0.125. Conclusion: Codelivery of pIL-12 with plasmid DNA can enhance the efficacy of immune responses and shift the type of immune responses.

Cloning of Porcine Lactoferrin Gene and Construction of Expression System in Recombinant Lactobacillus

Lactobacillus was selected as a bacterial carrier for expression of N-lobe of porcine lactoferrin （PLFN）. A pair of primers was designed with Oligo6.0 and used to amplify PLFN gene. It was in accordance with the characters of translational fusions from gene and expression vector plasmid. A 1 077 bp fragment of the gene from PLF was cloned from mammary gland tissue of the lactating sow on the third day by RT-PCR; the gene was connected with the vector plasmid pPG612.1 and transformed into the host strain JM109. The recombinant expression vector plasmid pPG612-PLFN was created and identified by using plasmid extraction, PCR, restriction enzyme digestion and sequence analysis. The recombinant plasmid was transformed into Lactobacillus casei ATCC393, Lactobacillus plantarum KLDS 1.0344, Lactobacillus paracasei KLDS 1.0652 and Lactobacillus pentosus KLDS 1.0413 by electroporation, and produced the recombinant strains of pPG612-PLFN/L, casei, pPG612-PLFN/L, plantarum, pPG612-PLFN/ L. paracasei and pPG612-PLFN/L, pentosus, respectively. The results indicated that PLFN gene had inserted into the expression vectors and achieved multiple Laetobacillus expression systems. It electes the base for the expression and production of recombinant porcine lactoferrin in Lactobaeillus

Overexpression of the mTERT gene by adenoviral vectors promotes the proliferation of neuronal stem cells in vitro and stimulates neurogenesis in the hippocampus of mice

We sought to construct the adenoviral vector carrying the gene encoding mouse telomerase reverse transcriptase（mTERT）,as well as detect its expression and effect on the proliferation of neuronal stem cells.mTERT was am-plified by RT-PCR and then the eukaryotic expression vector of pDC-EGFP-TERT was constructed.After DNA sequence analysis,we detected that there were 293 cells transfected with pDC-EGFP-TERT and helper adenovirus plasmid pBHG lox ΔE1,and three Cre using Lipofectamine 2000 mediation,named Ad-mTERT-GFP,to pack-age adenoviral particles.The Ad-mTERT-GFP was used to infect neuronal stem cells and then the expression and activity of mTERT were detected.In addition,Bromodeoxyuridine labeling test identified the impact of mTERT overexpression on proliferation of neuronal stem cells.The recombinant adenoviral vector confirmed that mTERT was successfully constructed.Overexpression of mTERT stimulated the proliferation of neuronal stem cells both in vitro and in vivo.mTERT overexpression via adenoviral vector carrying mTERT cDNA upregulated the ability of proliferation in neuronal stem cells.

Bioinformatics and Functional Analysis of High Oleic Acid-Related Gene GmSAM22 in Soybean [Glycine max (L.) Merr.]

High yield,high quality,stable yield,adaptability to growth period,and modern mechanization are the basic requirements for crops in the 21st century.Soybean oleic acid is a natural unsaturated fatty acid with strong antioxidant properties and stability.Known as a safe fatty acid,it has the ability to successfully prevent cardiovascular and cerebrovascular disorders.Improving the fatty acid composition of soybean seeds,can not only speed up the breeding process of high-quality high-oil and high-oleic soybeans,but also have important significance in human health,and provide the possibility for the development of soybean oil as a new energy source.Hence,the aim of this study was to analyze the high oleic acid elated gene GmSAM22 in soybean.In this research the soybean oleic acid-related gene GmSAM22 was screened out by Genome-wide association analysis,a 662 bp fragment was acquired by specific PCR amplification,and the pMD18T cloning vector was linked by the use of a seamless cloning technique.Bioinformatics analysis of the signal peptide prediction,subcellular localization,protein hydrophobicity,transmembrane region analysis,a phosphorylation site,protein secondary and tertiary structure and protein interaction analysis of the protein encoded by the SAM22 gene was carried out.The plasmid of the gene editing vector is pBK041.The overexpression vector was transformed from pCAMBIA3301 as the base vector,and overexpression vector were designed.Positive plants were obtained by genetic transformation by the pollen tube channel method.Fluorescence quantitative PCR was performed on the T2 generation plants to detect the relative expression levels in different tissues.Southern Blot was used to detect the presence of hybridization signal.Screening genes BAR,35S,and NOS in plants were identified by conventional PCR.10 seeds with high and low oleic acid content were chosen for quantitative PCR identification,and finally,the concentration and morphology of soybean fatty acids were identified by nearfar infrared spectroscopy.On 10 seeds with an upper and lower oleic acid content,a quantitative fluorescence analysis was done.In Southern blot hybridization,the SAM22 gene was integrated into the recipient soybean plant in hands of a sole copy.Fluorescence quantitative PCR appeared that the average relative expression of the SAM22 gene in roots,stems,leaves,and seeds was 1.70,1.67,3.83,and 4.41,respectively.Positive expression seeds had a 4.77%increase in oleic acid content.The level of oleic acid in the altered seeds was reduced by 4.13%when compared to CK,and it was discovered that the GmSAM22 gene could be a regulatory and secondary gene that promotes the conversion of stearic acid to oleic acid in soybean.There has not been a discussion of gene cloning or functional verification.The cloning and genetic transformation of the soybean SAM22 gene can effectively increase the content of oleic acid,which lays a foundation for the study of soybean with high oleic acid.

Construction and Identification of siRNA Expression Vector Targeting Nucleocapsid Protein N gene of PRRSV

[ Objective] The aim of this study was to investigate the construction and identification of siRNA expression vector targeting nucleocapsid protein N gone of PRRSV. [Method] Three siRNA oligonucleotides targeting nucleocapsid protein N gone sequence of PRRSV were designed or synthesized, and then inserted into CMV promoter downstream to clone into pSilencer 4,1 -CMV eukaryotic expression vector. The recombinant expression vector was identified by enzyme digestion and DNA sequencing. [ Result] The results showed that the siRNA interference recombinant plasmid vector pSilencer-N targeting nucleocapsid protein gone expression had been successfully constructed. [ Conclusion] This study lays a foundation for studies on the controlling PRRSV by RNA interference technique .

Construction of Double Cross-over Expression Vector for Chloroplast Multicistron in Brassica napus L.

[Objective] This study aimed to construct Brassica napus chloroplast multi- cistron double cross-over expression vector, to lay the foundation for the genetic engi- neering research of Brassica napus chloroplast. [Method] Two primers were designed based on the known Brassica napus chloroplast DNA sequences AF267640 and Z50868 in GenBank. By using PCR method, two Brassica napus L. chloroplast DNA fragments were obtained, which were named RbcL and ACCD. The two Brassica na- pus chloroplast DNA homologous fragments were then cloned into plasmid pMD18-T to obtain recombinant plasmid pHBM715. Tandem expression cassette harboring spectinomycin-resistant gene aadA, mannanase gene man and green fluorescent pro- tein gene gfp was cloned into the plasmid pHBM715, thereby constructing Brassica napus chloroplast multicistron double cross-over expression vector pHBM716, which was transformed into Escherichia coil for expression and identification. [Result] Plate qualitative analysis was conducted for the functional identification of expression cas- sette in the constructed Brassica napus chloroplast multicistron double cross-over ex- pression vector, results showed that the three genes of the same multicistron were all expressed in E. coil [Conclusion] This study successfully constructed Brassica napus chloroplast multicistron double cross-over expression vector, which laid the foundation for the genetic engineering of Brassica napus chloroplast.

Construction of Recombinant Retroviral Vector Containing HIV-1 Tat Gene and Functional Detection of Expressed Tat in Target Cells

Objective: To construct recombinant retroviral vector containing HIV-1 Tatgene and evaluate the junction of the expressed Tat in target cells. Methods: HIV-1 Tat_(101) genewas recovered from pEV plasmid by Hind Ⅲ digestion and cloned into expression plasmid LZESpBMN-Z toconstruct recombinant retroviral expression plasmid named LZRS-Tat_(101). Using the method ofcalcium phosphate, the construct of LZRS-Tat_(101) was then transfected into packaging cell linesPhoenix (ΦNX) which contained env and gal genes encoding structural proteins and pol gene codingfor 3 enzymes ( reverse transcriptase, protease and integrate) essential for retroviral integrationand replication . The stable transfected cell lines was obtained using puromycin to screen for morethan 3 days. Then, immunohistochemical (IHC ) staining was carried out to detect the expressionlevel of Tat_(101) protein in both transiently and stably trancfected ΦNX, respectively. Thesupematants containing recombinant virus collected from transient and stable transfected cells wereemployed to infect 293 cells, respectively, and the expressed Tat in 293 cells was tested by Westernblot. Meantime, the supematants of infected 293 cells was further added to HL3T1 cells which wereHela cell lines containing an HIV-1-LTR/CAT reporter construct to establish a co-culture system.After co-culture for 72 hours, the protein was extracted from HL3T1 cells and used for CAT activityassay. Results: After LZRS- Tat_(101) was transfected into ΦNX, the amount of expressed Tat intransient transfection cells was significantly higher than that in stable transfection cells; Tatcould be detected not only in 293 cells but also in the supematants from 293 cells culture, and Tatin the supematants could activate HIV-1 LTR promoter in HL3T1, resulting in high 'expression of CATlocated at the downstream of LTR. Conclusion: The construct of recombinant retrovirus LZRS-Tat_(101) could express Tat protein in target cells and the expressed Tat was functionally activeand can really exhibit the ability to activate transcription.

Construction of Plant-based Expression Vector of Polyphosphate Kinase Gene

[Objective] The aim was to construct the fusion expression vector of polyphosphate kinase（PPK） and green fluorescent protein（GFP） genes.[Method] In this study,the primers were designed based on PPK gene sequence（L03719） of E.coli DH5α in Genbank.Genomic DNA of E.coli DH 5α was extracted as template for the amplification of PPK gene by PCR method.By using In-Fusion@ HD Cloning Kit,the PPK gene was directionally cloned into NcoI site of the pCAMBIA1302 vector.[Result] Sequencing results showed that the 2.0 kb long fragment of PPK gene was inserted into the plant-based expression vector pCAMBIA1302 in front of GFP gene.[Conclusion] The fusion expression vector of PPK and GFP genes were successfully constructed.

Construction of Plant Virus Expression Vector pClYVV/CP/W and Expression of GFP

[ Objective] The study was to report the construction of plant virus expression vector pCIYVV/CP/W and the expression of green fluorescent protein（GFP） with pCIYVV/CP/W, and to develop effective plat virus vector for plant bioreactor to produce useful protein. [ Method] A section of multiple cloning sites among NIb/CP genes in pCIYVV genome and deoxyribonucleotide polylinker of cleavage recognition sequence containing viral protease Nla were cloned with infectivity full-length cDNA of clover yellow vein virus （CIYVV）, and pCIYVV/CP/W vector was constructed, GFP gene was inserted into pCIyVV/CP/W to construct the pCIYVV/CP/W/GFP vector. The transcription situation of recombinant virus clone was detected by RT-PCR, and targeted gene products expressed by recombinant virus clone were detected with western blot （WB）. [Result] The broad bean seedling inoculated with pCIYVV/CP/W/GFP expressed the same symptom as wild type CIYVV, morbidity was of 100%, the result showed that recombinant virus clone pCIYVV/CP/W/GFP didn＇t suppress, insertion of foreign gene didn＇t destroy the open reading frame of pCIYVV/CP/W. Foreign gene can keep living in F, progeny virus genorne steadily, recombinant virus clone pCIYVV/CP/W/GFP could steadily express GFP in progeny virus at least.[ Conclusion] The useful plant virus vector was provided for useful protein expressing.

Construction and Preliminary Identification of Eukaryotic Expression Vector of Cryptosporidium parvum miR-2980

[Objective] This study aimed to construct and preliminarily identify the eu- karyotic expression vector of Cryptosporidium parvum miR-2980. [Method] The cp-miR- 2980 precursor was amplified from C. parvum genomic DNA and cloned into pMD18- T vector. The amplified precursor was then subcloned into pVAX I vector and identi- fied with restriction endonuclease digestion and sequencing. The recombinant plasmid pVAX-miR2980 was transfected into HCT-8 cells. Total RNA was extracted and the expression of cp-miR-2980 was evaluated by RT-PCR detection. [Result] The results showed that the recombinant eukaryotic expression vector pVAX-miR2980 was suc- cessfully constructed, which can express cp-miR-2980 in HCT-8 cell. [Conclusion] This study laid the foundation for further exploring the biological function of cp-miR-2980.

Construction of Plant Expression Vector for Recombinant Human Epidermal Growth Factor (hEGF)

ObjectiveThis study aimed to construct plant expression vector for recombinant human epidermal growth factor （hEGF） and further to provide a basis for the expression of hEGF in peanut hairy root system. MethodAccording to the hEGF sequence in GenBank, hEGF was synthesized artificially; subsequently, hEGF gene was ligated with green fluorescent protein （GFP） gene, and their ligation product was then amplified with primers flanked with corresponding endonuclease cleavage sites, followed by double digestion by Sal I and EcoR I of the amplified products; next, pRI 101 AN DNA was extracted and digested by both Sal I and EcoR I; susequently, the digestion products of hEGF and GFP ligation fragment by Sal I and EcoR I and the digestion products of pRI 101 AN plasmid DNA by Sal I and EcoR I were ligated, and their ligation product was transformed into Escherichia coli XL10-Gold, followed by extraction of DNA from the recombinants exhibiting green fluorescence, which was then identified by enzymatic digestion and PCR, and the verified recombinant plasmid DNA was named pBZG101. ResultHuman epidermal growth factor gene （hEGF） and green fluorescent protein gene （GFP） were successfully ligated, and their ligation fragment was successfully ligated to pRI 101 AN DNA, finally with the acquirement of the plant expression vector for recombinant human epidermal growth factor-（pBZG101）. ConclusionThe plant expression vector for recombinant human epidermal growth factor-（pBZG101）- was successfully constructed in this study.

Sequence Analysis of Transcription Factor AtWRKY35 and Construction of Prokaryotic Expression Vector

As members of a super gene family, WRKY transcription factors are widely distributed in higher plants. ln this study, bioinformatic analysis of WRKY35, a member of the WRKY gene family, was carried out. Results indicated that tran-scription factor WRKY35 harbors a WRKYGQK core domain and a Cys2His2 or Cys2His/Cys zinc finger in the 5’ end without transmembrane domain. After PCR amplification and restriction digestion, WRKY35 gene fragment was ligated to prokaryotic expression vector PET28. This study provided basis for expression anal-ysis of WRKY35 protein and subsequent functional identification of WRKY35 gene.

Construction of Maize Universal Expression Vector PGM-35Sbar and Maize Transformation

[Objective] The paper was to construct maize universal expression vector, in order to provide basis for using transgenic methods to improve abiotic stress tolerance of maize. [Method] Based on the transformation of existing pGreen0229 plant expression vector, phosphinothricin-resistant selectable marker-bar gene driving by CaMv35S promoter was constructed, which could be used to connect target gene of maize expression vector PGM-35Sbar, and transform Ji444 maize inbred lines by pollen tube pathway. [Result] The universal expression vector for PGM-35Sbar maize had been successfully constructed. When the maize plants were transformed, 14 resistant plants were obtained, and 12 plants were identified to be positive plants by PCR detection. [Conclusion] The study provided basis for rapid construction of maize expression vector containing specific target gene.

Cloning of H6H Gene from Atropa belladonna and Construction of the Efficient Plant Expression Vector

[Objective] The aim was to clone H6H gene from Atropa belladonna and construct an efficient plant expression vector.[Method] The coding sequence of H6H（Hyoscyamine 6β-hydroxylase）was cloned from Atropa belladonna with RT-PCR.Then,the sequence was subcloned into the reconstructed plant binary expression vector p2301 to construct the recombinant vector p2301-H6H,which was then introduced into Agrobacterium tumefaciens strain LBA4404 and Agrobacterium rhizogenes strain C58C1,respectively.[Result] The engineering bacteria p2301-H6H-LBA4404 and p2301-H6H-C58C1 which could be directly used in genetic improvement were obtained.[Conclusion] The present research provided basis for the increasing of alkaloid content of Atropa belladonna by plant genetic engineering technology.

Construction of OsWRKY17 Specific Expression Vector in Rice

[Objective] To study the physiological biochemical characteristic of Os- WRKY17 in rice and identify the subcellular location of OsWRKY17. [Method] The primer of the OsWRKY17 gene was designed according to the full-length sequence of OsWRKY17 in Genbank and was cloned by RT-PCR. The cloned fragment was then recombined with the green fluorescent protein gene of plasmid vector pBinGFP. The recombinant plasmid pBinGFP-OsWRKY17 was transformed into Arabidopsis through Agrobacterium tumefaciens strain GV3101. [Result] Colony PCR and diges- tion identification proved that the plant expression vector pBinGFP-OsWRKY17 was successfully constructed by the fusion of OsWRKY17 and GFP, and the expression vector was successfully transformed into the genome of Arabidopsis, there by ob- taining a resistant plant. [Conclusion] The construction of OsWRKY17 expression vector established the foundation for study on the physiological the biochemical char- acteristics of QsWRKY17.

Construction of Eukaryotic Expression Vector for Pig Ghrelin Gene

[Objective] This study aimed to investigate the functions of transgenic growth related gene in pig growth. [Method] A pair of primers containing Nhe I and Hind Ⅲ restriction sites were designed by referring to the pig Ghrelin mRNA sequence published in Genbank. Total RNA was extracted from the small intestine tissue of 13/17 Robertson translocation heterozygous pig, and then was purified and used as the template in later RT-PCR reaction to amplify the full-length pig Ghrelin gene. The correct pig Ghrelin gene fragment was cloned into the pMD19-T simple vector for sequencing analysis. The obtained full-length cDNA of pig Ghrelin gene fragment was digested with both Nhe I and Hind Ⅲ, and then was linked into the eukaryotic expression vector pEGFP-N1 to obtain the recombinant plasmid pEGFPGhrelin. The recombinant plasmid was transected into the fibroblast cells to detect the fluorescence labeled gene expression. [Result] The nucleotide sequence extracted from 13/17 Robertson translocation heterozygous pig was the same as expected; and the eukaryotic expression vector pEGFP-Ghrelin was successfully constructed. [Conclusion] The eukaryotic expression vector constructed in this study can be further used in research on transgenic pigs, but also lays foundation for research on the regulatory mechanism of Ghrelin gene.