Cloning of a flower-specific expression promoter from Arabidopsis thaliana and its plant expression vector construction

Given the sequence of Chs gene promoter from Arabidopsis thaliana reported in GenBank （AF248988）, a pair of specific PCR primers was designed with the Primer Premier 5.0 software. PCR products of about 0.5 kb were successfully amplified with the genome DNA of A. thaliana as a DNA template and Taq polymerase as DNA polymerase. The purified PCR products were ligated to the pMD18-T vector. The sequencing result showed that the Chs promoter from A. thaliana was 531 bp long. Sequence alignment analysis based on the DNAMAN software revealed that the sequence similarity between the cloned promoter and target promoter （AF248988） was up to 100%. Online PLACE analysis indicated that the Chs promoter contained cis-elements such as TATA-box, CAAT-box, pollen-box, G-box, ACGT-containing element, R response element, Myb recognition element and TACPyAT-box. At the same time, a plant expression vectorpAtChs：：GUS which fused the Chs promoter and the marker gene GUS was successfully constructed.

Construction of Plant Expression Vector for Hand,Foot and Mouth Virus EV71-VP1 Gene and Its Expression in Tomato

EV71-type virus is one of the main pathogens causing the occurrence of hand,foot and mouth disease(HFMD),and VP1 protein,a factor that directly determines the antigenicity of the virus,has been isolated.The tomato was selected as a bioreactor for the production of an edible EV71 vaccine designed for the VP1 capsid protein.Using molecular biology techniques,the fusion gene EV71-VP1 was cut from vector PGEX-4T-2,a vector containing the p2300-EV71 gene with CaMV35S promoter and TL regulatory elements was constructed,and the hypocotyl and cotyledons of tomato were transformed using Agrobacterium(EHA105)-mediated method,screened,elongated and rooted,and finally 20 resistant tomato plants were obtained.Five transgenic positive seedlings were obtained by digestion and PCR assay,among which three plants were detected by RT-PCR to be capable of transcriptional translation at the RNA level.The experimental results aimed to explore new material support for the preparation of transgenic plant oral vaccines against EV71 infection and provide a theoretical basis for accelerating the development of transgenic plant vaccines in the future.

Construction and Identification of a Goat Pox Virus Transfer Vector to Express Peste des Petits Ruminants H gene

[Objective] This study was to develop a live vector vaccine of goat pox virus of Peste des petits ruminants(PPR). [Method] Using PCR amplification technique, PPR H gene was obtained, then ligated into pGEM-T easy vector; the recombinants were digested by Nhe Ⅰ and Hind Ⅲ, and ligated into pEGFP-N1-P7.5, yielding the recombinant vector pEGFP-N1-P7.5-H; next the expression cassette EGFP-N1-P7.5-H was first released from recombinant vector pEGFP-N1-P7.5-H by double digestion of Hind Ⅲ and Nhe Ⅰ and ligated into pUC119-TK that was digested by Kpn Ⅰ, yielding the transfer vector pUC119-TK-EGFP-P7.5-H. [Result] Identification and double enzyme digestion showed that the transfer vector pUC119-TK-EGFP-P7.5-H was correctly constructed. From the transfer vector transfected BHK-21 cells which infected GTPV AV41, specific fluorescence was observed at 48th h of transfection. [Conclusion] The construction of goat poxvirus live vector laid a foundation for the live vector vaccine of PPR vaccine.

Construction of a RNA Interference Vector of Brassica napus L.Pyruvate Kinase Gene

A predominantly expressed gene, pyruvate kinease （PK） gene, control ing oil accumulation, had been identified in previous study. To construct a PK RNAi vector, a 498-bp target PK gene sequence was amplified and transferred into the pEASY-T1vector. Subsequently, the target DNA fragments were cut off by enzymes Not I and Xho I and directional y inserted into plant RNAi platform vector pHurricane, a newly developed RNAi vector in our lab, to form the PK inverse repeats. The PK inverted repeats fragment was then cloned into a modified vector pCAMBIA1390, driven by a rapeseed seed-specific promoter napin. Restriction enzyme digestion verified the successful construction of RNA interference vector. The PK RNAi con-struction wil lay a foundation for study in the function of PK in oil accumulation and metabolism in rapeseeds.

Construction of Expression Vector for Porcine Gastrin-releasing Peptide Fusion Protein

[Objectives]This study was conducted to obtain porcine gastrin-releasing peptide(GRP)fusion protein.[Methods]The constructed pET32a(+)-GRP plasmid was transformed into Escherichia coli BL21(DE3)competent cells to obtain the pET32a(+)-GRP-BL21(DE3)fusion protein expression strain,which was induced with 0.5 mM IPTG at 25℃and 150 r/min for 12 h,and the His-tagged GRP fusion protein was detected by SDS-Page gel electrophoresis and Western Blot.[Results]After optimizing the IPTG-induced expression conditions,it was confirmed that the porcine GRP fusion protein was obtained,and the porcine GRP fusion protein was soluble,stable and highly active.[Conclusions]This study lays a foundation for the subsequent preparation of anti-pig GRP antibodies.

Cloning of Broad-spectrum Anti-disease NPR1 Gene with RT-PCR and Construction of Its Protein Expression Vector

[Objective] It is to clone broad-spectrum anti-disease gene NPR1 and to construct its protein expression vector.[Method] First to extract total RNA of Arabidopsis thaliana and design relevant primers,and then the method of reverse transcription PCR was adopted to clone.With the method of enzyme digestion and ligation,this gene will be directed into protein expression vector.[Result] After relevant testing,NPR1 was inserted into vector pMXB10 to obtain pMXB10-NPR1 protein expression vector.[Conclusion] Protein expression vector including NPR1 was successfully constructed.

Construction of plant expression vector of Pseudopleuronectes americanus antifreeze protein gene

The Pseudopleuronectes americanus antifreeze protein gene was synthesized and control sequences were added such as 35S promoter and nos terminator that can facilitate the transcription and Ω sequence and Kozak sequence that can improve the expression in translation level, the high expression cassette of antifreeze protein was constructed. This cassette was connected to pBI121.1 and finally got the high expression vector pBRTSAFP introduced into the maize callus. The expression of gus gene that linked to the antifreeze protein gene was detected, and the results was that the gus gene can express strongly and instantaneously.

Construction of RNAi Expression Vector against Riboflavin Synthase Gene

[Objective] This study aimed to construct RNAi expression vector against r/boflavin synthase （RS） gene. [Method] By using the primers designed based on RS gone coding sequence that was screened from Arabidopsis cDNA library, the 476 bp cDNA fragment of RS was amplified from pGADTT-RS recombinant plasmid, and then cloned into pUCm-T vector to obtain pUCm-RS. Two RS fragments （476 bp） were obtained through digesting pUCm-RS with restriction enzymes PstI/BamHI and PstI / Xhol, and then respectively connected into vector pBSSK-in to form pBSSK-RS-in-RS, in which the two RS fragments were inverted re- peats. Finally, the transform unit RS-intron-RS, got by digesting vector pBSSK-RS-in-RS with Sac I and Kpn I, was ligated into expression vector pCAMBIA1301 to obtain the RS gene silencing vector. [ Result] The restriction enzyme digestion sequencing analysis proved that the RS gene silencing vector was successfully con- structed. [ Conclusion] This study may provide a basic material for further studies on the bio-function of RS gene and the mechanism of signal transduction induced by HpaGxoo in plant.

Construction of Prokaryotic Expression Vector for At4g12500 Gene of Arabidopsis

[ Objective] This study aimed to construct the prokaryotic expression vector for Arab/dops/s At4g12500 gene. [ Method] Genomic DNA of wild-type Ar- abidopsis Cold） was extracted as the template for amplification of At4g12.500 gene with PCR technology. The target fragment was double-digested with BamH I and Xho I, and ligated with prokaryotic expression vector pET-28a. [Result] Prokaryotic expression vector pEI28a-At4gI2500 was successfully constructed and trans- formed into Escherichia coli expression strain BL21 （DE3）. [ Conclusion] This study laid the foundation for subsequent expression and functional analysis of At4g12500 gene.

Construction and Expression of Sugarcane UGPase cDNA Prokaryotic Expression Vector

UGPase （UDP-glucose pyrophosphorylase）, one of the primary enzymes concerned with carbohydrate metabolism, catalyzes the formation of UDPG. By inserting the UGPase cDNA fragment cloned from Saccharum officinarum into PQE-30, the prokaryotic expression vector of PQE-UGP was successfully constructed. Then the vector plasmid of PQE-UGP was transformed into host bacteria M 15 and the expression of target gene was induced by Isopropyl β-D-1-Thiogalactopyranoside （IPTG）. The research laid foundation for study on the prokaryotic expression of UGPase.

Construction of PBIB Desings by Using Subspace of Vector Space over Finite Fields

A new transitivity theorem of the general linear group GLn(Fq)is proved. A kind of new PBIB desings is constructed.

Construction of Cox7a2 fluorescent vector and its effect on cytochrome C oxidase activity in mouse Sertoli cell line TM4

Objective To construct Cox7a2 fluorescent vector and study its effect on cytochrome C oxidase ( COX) activity in mouse Sertoli cell line TM4. Methods The coding region of CoxTa2 was amplified from mouse Sertoli cell line TM4 by RT-PCR. PCR product was

Construction and identification of Fas-targeting siRNA-expressing plasmid

Objective: To study the therapeutic potential of Fas inhibition in different diseases, a Fas-targeting siRNA (small interfering)-expressing plasmid was constructed. Methods: The U6 promoter cassette and siFas (small interfering RNA that inhibit Fas expression) template sequence were obtained by PCR method. They were cloned into modified pcDNA3.1. The resultant plasmid pU6-siFas was transfected into P815 cells with lipofectin2000 and selected under G-418-containing culture medium. Fas inhibition in stably transfected cells was detected by immunocytochemistry. Results: The plasmid pU6-siFas efficiently reduced the expression of Fas and conferred G-418 resistance in P815 cells. Conclusion: The successful construction of the siRNA expressing plasmid will facilitate the application of RNA interference technique and lay the foundation for further study of Fas inhibition in the treatment of different diseases such as aplastic anemia and acute liver failure.

Identification of Brucella melitensis and Molecular Cloning of Its BP26 Gene into p ET24a(+) Vector

Brucella spp. are pathogenic to humans and domestic animal. Nowadays,there is no effective vaccine and control strategy in China. So,it is necessary to research effective vaccines for prevention and treatment of this disease. In order to deal with these,we isolated and identified the type of Brucella in Darhan Muminggan Joint Banner and Siziwang Banner of Inner Mongolia. Totally 26 samples of sheep blood which were positive in serological test,one sample of spleen from aborted and one sample of secretion from birth canal were isolated,and the 16 S r DNA genes of positive samples were sequenced. Phylogenetic analysis proved that there were four isolates similar to B. melitensis. As an important diagnostic antigens of B. melitensis,the BP26 gene was amplified. The BP26 gene was cloned into vector p ET24a( +) and conducted sequence analysis. The BP26 gene was 900 bp,with an open reading frame of 753 bp. The homology of BP26 gene with the vaccine strain M5 was 100%,and that with S2 and A19 vaccine strains was 99. 99%. These finding supported the development of BP26-based specific serodiagnostic test and vaccine for B. melitensis in China.

Cloning of BjNAC102 Promoter and Construction of Expression Vector in Brassica juncea

Transcription factor NAC102 plays an important role in the abiotic stress responses of plants.In this study,the promoter sequence of 3000 bp located in the upstream of the BjNAC102 gene was cloned from Brassica juncea‘Sichuan Yellow Seed’by using the homologous cloning method.The expression vector of the GUS gene driven by the BjNAC102 promoter was constructed by seamless cloning technology.The results showed that the sequence of the promoter of the BjNAC102 gene contained many cis-acting elements involved in light responsiveness,gibberellinresponsive element,and auxin-responsive element.It was speculated that BjNAC102 played an important role in the abiotic stress response in Brassica juncea.The expression vector of the promoter of the BjNAC102 gene was constructed,which layed a foundation for further studies of the expression pattern of the BjNAC102 gene in Brassica juncea.

Recombination and identification of human alpha B-crystallin

AIM: To recombine the human alpha B-crystallin(αBcrystallin) using gene cloning technology and prokaryotic expression vector and confirm the biological activity of recombinant human αB-crystallin. METHODS: Cloning the human αB-crystallin cDNA according to the nucleotide sequence of the human αBcrystallin, constructing the pET-28/CRYAB prokaryotic expression plasmid by restriction enzyme digestion method, and stably expressing transformed into the Escherichia coli(E. coli) DH5 alpha. The recombinant human αB-crystallin was purified by Q sepharose. By enzyme digestion analysis, Western blotting and sequencing, the recombinant human αB-crystallin was identified and the activity of its molecular protein was detected.RESULTS: Compared with the gene bank(GeneBank), the cloned human sequence of human αB-crystallin cDNA has the same open reading frame. Identification and sequencing of the cloned human αB-crystallin cDNA in prokaryotic expression vector confirmed the full length sequence, and the vector was constructed successfully. The E. coli containing plasmid pET-28/CRYAB induced by isopropyl-β-D-thiogalactoside successfully expressed the human αB-crystallin. Insulin confirmed that the recombinant human αB-crystallin has a molecular chaperone activity. CONCLUSION: The prokaryotic expression vector pET-28/CRYAB of recombinant human αB-crystallin issuccessfully constructed, and the recombinant human αBcrystallin with molecular chaperone activity is obtained, which lay a foundation for the research and application of the recombinant human αB-crystallin and its chaperone activity.