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.

Expression Vector Construction and Genetic Transformation of <i>Rosa rugosa β</i>-l,3-Glucanase Gene (<i>RrGlu</i>)

In order to lay a foundation for researching the function of Rosa rugose (R. rugosa) RrGlu gene, the RrGlu gene was amplified from the styles of R. rugosa “Tanghong”, a gene expression vector named PBI121-RrGlu was constructed and the vector was introduced into tobacco with the agrobacterium-mediated method. PCR results showed that the RrGlu gene was integrated into the tobacco genome.

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.

The Construction and Identification of Eukaryotic Expression Vector pEGFP-N1-hTERT

[Objective] The aim of this study is to construct eukaryotic expression vector pEGFP-N1-hTERT and observe its expression in eukaryotic cells.[Method]The eukaryotic expression vector pEGFP-N1-hTERT was constructed with pC1-neo-hTERT and pEGFP-N1 plasmids,and the accuracy of human telomerase reverse transcriptase(hTERT)gene fragment was confirmed by double enzyme digestion and DNA sequencing analysis.After transfecting pEGFP-N1-hTERT into rat fetal neural stem cells(NSCs),the protein localization of human telomerase reverse transcriptase were indirectly observed through green fluorescent protein in the cells,and the correctness of constructed pEGFP-N1-hTERT was certificated by RT-PCR and Western Blot analysis.[Result]The eukaryotic expression vector pEGFP-N1-hTERT had correct structure and could express in eukaryotic cells.[Conclusion]This study laid a foundation for the establishment of immortalized NSCs line in rats.

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.

Comparison of School Building Construction Costs Estimation Methods Using Regression Analysis, Neural Network, and Support Vector Machine

Accurate cost estimation at the early stage of a construction project is key factor in a project’s success. But it is difficult to quickly and accurately estimate construction costs at the planning stage, when drawings, documentation and the like are still incomplete. As such, various techniques have been applied to accurately estimate construction costs at an early stage, when project information is limited. While the various techniques have their pros and cons, there has been little effort made to determine the best technique in terms of cost estimating performance. The objective of this research is to compare the accuracy of three estimating techniques (regression analysis (RA), neural network (NN), and support vector machine techniques (SVM)) by performing estimations of construction costs. By comparing the accuracy of these techniques using historical cost data, it was found that NN model showed more accurate estimation results than the RA and SVM models. Consequently, it is determined that NN model is most suitable for estimating the cost of school building projects.

木薯MeMLO12基因克隆及其CRISPR-Cas9表达载体的构建

MLO基因是植物中特有的一类抗病性负调控因子,该基因突变导致植物产生广谱抗病性。本研究从木薯全基因组中克隆获得木薯MLO12基因的DNA和cDNA序列,并将其命名为MeMLO12。该基因全长3743 nt、编码区(ORF)全长1728 nt,具有一个完整的开放阅读框,含有15个外显子和14个内含子,编码586个氨基酸,蛋白的分子质量为67.2 kDa,等电点为8.85。该基因编码的蛋白定位在内质网膜上,无信号肽,在23~45、74~96、161~183、285~307、312~334、371~393、413~435 aa处形成7次跨膜结构域。qRT-PCR定量分析发现,受木薯黄单胞病菌侵染后,MeMLO12基因在木薯抗、感品种中的表达量存在明显的差异,参与木薯与黄单胞菌之间的互作,表现出负调控作用。选择该基因第11个外显子进行Snap Gene Viewer分析,获得了10 455条sgRNA的种子序列,从中选取3条靶序列约23 nt,碱基组成上3'末端含G结尾,将其构建到CRISPR-Cas9载体上,经验证,确认MeMLO12的3条靶序列已经成功构建到基因编辑载体上,将其命名为pSGR-Cas9-AT-MeMLO12载体。

植酸酶基因在嗜酸乳杆菌中的表达

植酸酶和嗜酸乳杆菌是重要的饲料添加剂,为获得产植酸酶的嗜酸乳杆菌菌株,克隆了大肠杆菌植酸酶基因,构建表达载体并转化嗜酸乳杆菌,对重组嗜酸乳杆菌进行发酵产酶并测定所产酶液的酶学性质,进一步研究重组嗜酸乳杆菌的耐酸性及植酸酶液的抗蛋白酶活性。结果表明:重组嗜酸乳杆菌发酵24 h植酸酶活性为984 U/mL,植酸酶的最适催化pH为4.5,最适催化温度为60℃。重组嗜酸乳杆菌在pH 2.0~4.5有一定的耐酸性,在pH 2.0条件下2 h的存活率为76.4%。植酸酶液用胃蛋白酶处理160 min后植酸酶液剩余85%的相对酶活,用胰蛋白酶处理160 min后剩余29%的相对酶活。上述研究结果为重组嗜酸乳杆菌后续应用研究提供了重要依据。

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