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.

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.

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.

Cloning of CBF3 Gene from Arabidopsis thaliana and Construction of Plant Expression Vector

[Objective] The aim was to clone CBF3 gene from Arabidopsis thaliana and construct plant expression vector pCAMBIA1301-Rd29A-CBF3.[Method] CBF3 gene and stress-inducible promoter Rd29A were amplified from the genomic DNA of A.thaliana for the construction of plant expression vector.[Result] Sequencing results showed that the cloned CBF3 gene had 750 bp,and showed 100% identity with the sequence published on GenBank.The promoter Rd29A had 1 425 bp,and showed 100% identity with the sequence published on GenBank.[Conclusion] Based on the binary vector pCAMBIA1301,the plant expression vector pCAMBIA1301-Rd29A-CBF3 was constructed successfully,which could materially improve the salt resistance,drought-tolerance,cold resistance of plants.

Cloning the Promoter of BcNA1 from Brassica napus and Fad2 Gene from Arabidopsis thaliana and Construction of the Plant Expression Vector

The upstream regulatory region of a seed specific gene was isolated from the genomic DNA of Brassica napus by PCR amplification. The cloned fragment contained 1755 nucleotides, and shared a sequence homology of 99.6% with the reported data. The coding region of oleic acid desaturase gene was then cloned from Arabidopsis thaliana. The sequencing analysis indicated that the sequence of the PCR product was just the same as reported before. In addition, the plant expression vector harboring the seed specific promoter and trans Fad2 gene was constructed.

Cloning of XET gene from Anthocephalus chinensis and its plant expression vector construction

A full-length cDNA sequence of xyloglucan endotransglycosylase gene （XET）, abundantly expressed in the cambium of Anthocephalus chinensis was cloned by conserved PCR, rapid-amplification of cDNA ends and by chromosome walking. Analytical results of the DNA sequence show that a 912 bp complete open reading frame （ORF） encoded a 303-amino acid protein was in the 1205 bp full cDNA sequence. The deduced amino acid sequence of AcXET, which contained the conserved specific EIDFE catalytic site sequence to XETs was homologous to the other known XET proteins. In order to study the gene function of AcXET and obtain transgenic plants, a plant expression vector pBIAcXET was constructed by recombinating the AcXET fragment from the cloning vector pMD19AcXET and the binary vector pBI121 between the XbaI and SmaI sites. The fragment ofAcXET gene was inserted between the CaMV 35S promotor and the coding region of the GUS gene in pBI121. The identification results show that the plant expression binary vector pBIAcXET was constructed successfully. These results lay the foundation for studying the molecular mechanism ofAcXET gene during wood formation.

Construction of Plant Expression Vector of Heat Shock Factor Gene AtHsfA1a from Arabidopsis

[ Objective ] Heat shock factors （HSFs） are the major transcription factors of eukaryotic heat shock responses. This study aims to investigate the adversity stress tolerance functions of Arabidopsis heat shock factor AtHsfAla, which has important significance for in-depth understanding of adversity stress tolerance mechanisms of plants and further utilization of heat shock factor genes. [Method] Genomic DNA of Arabidopsis was extracted with CTAB method and purified to obtain Arabidopsis DNA samples for in vitro site-specific recombination cloning （ Gateway cloning） to construct plant expression vector of heat shock factor AtHs- fAla. Firstly, donor vector pDONR 201/AtHsfAla was constructed based on attB and attP site-specific recombination method （BP reaction）, to identify E. coli transformants harboring correct sequence of AtHsfAla by sequencing; secondly, plant expression vector pBTWG2/AttlsfAla overexpressing Arabidopsis heat shock factor AtHsfAla was constructed based on attL and attR site-specific recombination method （LR reaction）, to screen E. coli transformants harboring target plasmid. [ Result] Plant expression vector of Arabidopsis heat shock factor gene AtHsfAla was constructed successfully. [ Conclusion] This study not only provided experimental materials for acquiring transgenic plants overexpressing heat shock transcription factor AtHsfAla, but also laid the foundation for further investigation of the diversity of adversity stress tolerance functions reanlated by HSFs.

Cloning and Bioinformatics Analysis of ZmERECTA-LIKE1 and Construction of Plant Expression Vector

[Objective] This study was conducted to clone and analyze ERECTA-LIKE1 gene in Zea mays by PCR and bioinformatics methods and to construct plant expression vector p Cambia3301-zm ERECTA-LIKE1. [Method] zm ERECTA-LIKE1(zm ERL1)gene was obtained using RT-PCR, and physical-chemical properties were analyzed by bioinformatics methods, including domains,transmembrane regions, N-Glycosylation potential sites phosphorylation sites, and etc. [Result] Bioinformatics results showed that zm ERL1 gene was 2 169 bp, which encoded a protein consisting of 722 amino acids, 11 N-glycosylation potential sites and 42 kinase specific phosphorylation sites. According to CDD2.23 and TMHMM Server v. 2.0 software, there were leucine-rich repeats,a PKC domain and a transmembrane region in this protein. The theoretical p I and molecular weight of zm ERL1 encoded protein was 6.20 and 79 184.8 using Compute PI/Mw tool. Furthermore, we constructed the plant expression vector p Cambia3301-zm ERECTA-LIKE1 by subcloning zm ERL1 gene into p Cambia3301 instead of GUS. [Conclusion] The results provide a theoretical basis for the application of zm ERL1 gene in future study.

Generating Marker-Free Transgenic Tobacco Plants by Agrobacteriummediated Transformation with Double T-DNA Binary Vector

We have developed a 'double T-DNA' binary vector system for generating selectable marker-free transgenic plants by Agrobacterium-mediated transformation. The 'double T-DNA' binary vector pDLBRBbarm which carried two independent T-DNAs, one containing a selectable marker neomycin phosphotransferase (nptII) gene and the other a bargene, was constructed. Transgenic tobacco (Nicotiana tabacum L.) plants were then produced by Agrobacterium-mediated transformation with this vector. Frequency of the primary transformants co-integrated with npt II gene and bar gene was 59.2%. Segregation of two T-DNA regions was found in 3 out of 4 T-1 lines from co-transformed T-0 plants with nptII and bar PPT-resistant and kanamycin-sensitive plants were in approximate 19.5% of the T-1 plants. The result indicated that this 'double T-DNA' vector system could be a workable approach to generate transgenic plants free from selectable marker genes. Co-transformation of nptII gene and bar gene to plants with mixtures of Agrobacterium tumefaciens strains containing single T-DNA vectors was also tested. Frequency of co-transformed plants was 20.0%-47.7% and relatively low as compared with that of 'double T-DNA' vector system.

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.

大白菜BrCYP83B1基因的克隆及表达分析

【目的】细胞色素P450家族是十字花科植物硫苷合成重要的酶系,其中CYP83亚家族主要参与核心结构的合成,旨在探究大白菜(Brassica rapa ssp.pekinensis)CYP83B1基因的功能。【方法】利用RT-PCR技术克隆BrCYP83B1基因,通过生物信息学软件分析其编码蛋白理化性质、同源性及启动子顺式作用元件,利用RT-qPCR技术分析BrCYP83B1的表达模式,并构建其植物超表达载体。【结果】BrCYP83B1 cDNA序列全长为1500 bp,编码499个氨基酸,编码蛋白属于细胞色素P450超家族,主要定位于细胞质,二级结构主要由α-螺旋和无规则卷曲构成,与甘蓝型油菜、青花菜的CYP83B1蛋白具有较高的同源性。启动子分析表明,该基因启动子区域包含水杨酸、脱落酸及茉莉酸甲酯等激素响应的顺式作用元件,说明BrCYP83B1基因表达可能受激素调控。RT-qPCR分析结果表明,BrCYP83B1基因在大白菜的根、茎、叶、花和果中均有表达,且以叶中的表达量最高;茉莉酸甲酯够显著促进该基因的表达,而水杨酸处理对其表达具有一定的抑制作用,脱落酸处理下基因先上调后又下调。【结论】BrCYP83B1可能参与大白菜对激素的响应调控。

DREB基因双T-DNA植物表达载体的构建及验证

W 34基因经 pGEM T EASY载体 ,克隆到 pBDREB载体EcoRI位点 ,得到在Amp平板上生长的转化子 pBW 34-DREB质粒 ;质粒pBW 34-DREB、pSPROK、pBlueks分别经PstI/KpnI、KpnI/EcoRI、PstI/EcoRI双酶切 ,三片段连接构建成中间表达载体 pBWD Tnos;再用SmaI/EcoRV双酶切 pBWD Tnos,将回收的DREB基因的完整的表达结构元插入到经SmaI酶切的质粒载体 pCDMAR Hyg中 ,构建成双T DNA植物表达载体 pCDMAR pWDT Hyg ,大小为 13 5 9kb ,经酶切鉴定 ,证明构建的载体与预期设计的一致。将此双T载体用农杆菌介导法转化玉米胚性愈伤组织 ,期望得到无选择标记的安全的转基因植物。

At2基因双T-DNA植物表达载体的构建

以甜瓜品种‘MR-1’为试材,采用PCR技术,扩增出了At2基因并在两端添加BamHⅠ和SacⅠ酶切位点。结果表明:在NCBI上进行BLAST显示其氨基酸序列与GeneBank中已公布的At2基因的同源性为99%;经DNAMAN软件分析,核苷酸序列同源性为99.43%。将其连接在中间表达载体G-pPTN133上,得到替换了GUS基因的中间载体A-pPTN133。A-pPTN133再经ScaⅠ酶切后,插入到经ScaⅠ酶切的pPTN133^-中,构建成双T-DNA植物表达载体At2-pPTN133。经酶切和PCR鉴定证明了所构建载体的正确性。

番木瓜β-Gal基因RNAi双T-DNA植物表达载体的构建及其遗传转化的初步研究

克隆了番木瓜(Carica papaya L.)果肉的细胞壁水解关键酶β-半乳糖苷酶(β-GAL)基因保守区,将其反向重复插入载体pKANNIBAL,构建RNAi中间表达载体pKAN/RG,将其上的发夹结构取代经改造的载体pCAMBIA1300上hptⅡ基因,构建中间表达载体p1300-/MFRG,分离单T-DNA区段,与载体pCAMBIA2301构建RNAi双T-DNA植物表达载体p2301/TTRG。酶切分析和PCR检测表明,p2301/TTRG已被成功导入农杆菌EHA105。通过遗传转化,初步获得了GUS染色呈阳性且具Kan抗性的番木瓜胚性愈伤组织。

CTM2-GFP转基因拟南芥构建及细胞定位研究

通过筛选突变体库发现的镉耐受突变体植株可能参与了植物Cd胁迫的调控。文章通过同源重组方法构建pART27-CTM2-GFP载体,并将pART27-CTM2-GFP重组质粒转入农杆菌GV3101,采用浸花转染法转入野生型拟南芥植株;通过抗性筛选和聚合酶链式反应(polymerase chain reaction,PCR)鉴定获得CTM2-GFP转基因阳性植株,进行抗性分离鉴定获得纯合转基因植株;最后通过qPCR检测CTM2基因表达,筛选最佳的CTM2-GFP转基因株系;通过观察CTM2-GFP转基因植株中绿色荧光蛋白(green fluorescent protein,GFP)的荧光发现CTM2为细胞膜定位蛋白。研究结果为深入研究CTM2基因在调控植物镉耐受分子机制中的作用提供了遗传材料支撑。

植物防龋疫苗融合基因表达载体中选择标记基因的去除

背景:出于对转基因植物的食用安全和生态安全的考虑,标记基因的存留是影响转基因植物的首要安全问题。目的:基于免疫防龋原理,课题组针对表面蛋白和葡糖基转移酶这2种致龋毒力因子,构建植物防龋疫苗融合基因表达载体pCAMBIA-E8-APB-DOCK8,为转基因植物疫苗的研发提供基础。方法:该研究运用DNA重组技术,通过DNA片段分离、连接、转化、克隆子检测、测序等系列步骤,将植物防龋疫苗融合基因表达载体pCAMBIA-E8-APB-DOCK8中的选择性标记基因Km和GUS去除。结果与结论:标记基因去除效率达99%,该研究为免疫防龋的转基因植物疫苗的安全生产奠定了良好的实验基础,也为其它植物疫苗的载体构建提供思路。

LFY基因双T-DNA植物表达载体的构建

LFY基因是从从野生型拟南芥中克隆出的一个与调控花分生组织启动相关的基因,它有促进植物提早开花的作用。用PCR方法从克隆载体上扩增出LFY基因并在两端添加XhoⅠ酶切位点,将其连接在用XhoⅠ切除了hyg基因的pCAMBIA1301表达载体上,得到去除选择标记的LFY基因植物表达载pCAM-BIA1301-LFY。质粒pCAMBIA1301经HindⅡ/EcoRⅠ双酶切补平自连后得到去除多克隆位点的质粒pCAMBIA1301-,再经SacII/SphI双酶切后插入到经SacⅡ酶切的pCAMBIA1301-LFY中。建成双T-DNA植物表达载体pCAMBIA1301-LFY-hyg-,经酶切鉴定证明了所构建载体的正确性。将此双T-DNA载体用农杆菌介导法转化菊花叶片,期望得到无选择标记含LFY基因的转基因菊花。

多基因双T-DNA植物表达载体的构建及拟南芥转化

多基因植物表达载体用于植物遗传转化是培育具有多种优良品质作物的有效策略.双T-DNA系统是实现筛选完成后选择标记基因删除的一种简便可行的方式.为培育高度抗逆或去除标记基因的农作物,构建了多基因双T-DNA植物表达载体2T-bbgdD,其中含有一个抗除草剂基因bar,3个抗逆相关基因(DREB1A,Na+依赖性Pi转运体基因(d5),betA)和一个报告基因gfp.利用农杆菌介导法将该载体转入拟南芥,获得了多基因共转化及去除标记基因的转基因拟南芥.可将此植物表达载体进一步用于作物的遗传转化.

Construction of plant expression vectors carrying glnA gene encoding glutamine synthetase and regeneration of transgenic rice plants

The glnA gene encoding glutamine synthetase (GS) was amplified from Azospirillum brasilenseSp7 with PCR technique.The amplified 1.4-kb DNA fragment flanked with a BamH Ⅰ site at each end wascloned into EcoR V site of Bluescript-SK vector.A recombinant plasmid pGSJ1 containing this 1.4-kb DNA frag-ment was selected by restriction digestion analysis.The sequencing data also confirmed that the amplified 1.4-kbDNA fragment was undoubtedly the glnA gene of A.brasilense Sp7.Then the 1.4-kb BamH Ⅰ fragment was ex-cised from pGSJ1.A glnA plant expression vector pAGNB92 with rice actin 1 (Act1) promoter was constructedby using colony in situ hybridization to screen positive clones,and 3 rounds of ligation and transformation wereperformed.Protoplasts isolated from rice (Oryza sativa,L.Japonica) cell suspension line (cv.T986) weretransformed with the glnA plant expression vector pAGNB92 carrying neomycin phosphotransferase Ⅱ (NPT Ⅱ)gene by PEG fusion or electroporation.G418~ calli were used to detect NPT Ⅱ enzyme activity.The resultsshow that G418~ calli possess high positive hybridization signal with the frequency of 37%.The regeneratedG418~NPTII^+ rice plants were used for PCR amplification of glnA gene,and a 1.4-kb DNA fragment was ampli-fied from glnA-transgenic rice plants (R0 generation).The results of Southern blot hybridization prove that the1.4-kb DNA fragment amplified from the total DNA of glnA transgenic rice plants is indeed the glnA gene of A.brasilense Sp7.Northern blot hybridization was carried out using the same glnA gene as probe.The glnAgene was expressed in the transgenic rice plants.Bioassays also confirmed that the glnA transgenic rice plantsgrew much better than that of the control plants under a condition with nitrogen poor source (0.75 mmol/L).

A Nimble Cloning-compatible vector system for high-throughput gene functional analysis in plants

Plant expression vectors are essential tools for gene functional analysis and molecular plant breeding.The gene of interest is transferred to the vector by molecular cloning technology.Nimble Cloning is a newly developed molecular cloning method with the advantages of simplicity,efficiency,and standardization.In this study,we developed a"pNC"vector system that contains 55 Nimble Cloning-compatible vectors for functional analysis of genes in plants.These vectors contain the NC frame flanked by unique adapters for one-step and standardized Nimble Cloning.We demonstrate that the pNC vectors are convenient and effective for the functional analysis of plant genes,including the study of gene ectopic expression,protein subcellular localization,protein-protein interaction,gene silencing(RNAi),virus-induced gene silencing,promoter activity,and CRISPR-Cas9-mediated genome editing.The"pNC"vector system represents a high-throughput toolkit that can facilitate the large-scale analysis of plant functional genomics.