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 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.

Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was amplified by RT PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.

A versatile cloning vector facilitates target geneexpression in prokaryotic and eukaryotic cells

Objective： To facilitate manipulation of gene expression in different host cells, we used pEGFP-N1 as backbone to construct a versatile vector that can drive foreign gene expression in prokaryotic and eukaryotic cells. Methods： A cloning and expression vector, pEGFP-NI-lac, was constructed by inserting the prokaryotic lac promoter of pUC 19 into the eukaryotic expression vector, pEGFP-N1, between the eukaryotic PCMV promoter and enhanced green fluorescent protein （EGFP） open reading frames. To assess the function of pEGFP-NI-lac, the nucleotide sequence encoding the hepatitis C virus （HCV） core protein was cloned into the multiple cloning sites. Western blotting analysis was used to detect the expression of the HCV core protein in Escherichia coli DH5a and HepG2 cells. Results： Restriction enzyme digestion and sequence analysis indicated that pEGFP-NI-lac was successfully constructed and the HCV core gene was cloned into this vector. The Western blotting results showed that pEGFP-NI-lac promoted expression of HCV core gene in prokaryotic E. coli DH5a and eukaryotic HepG2 cells. Conclusion： The pEGFP-NI-lac vector has been successfully constructed and functions in both prokaryotic and eukaryotic cells. The EGFP reporter can be used as an insert-inactivation marker for clone selection or as an expression tag. This vector can be used for cloning and expression of genes in both prokaryotic and eukaryotic cells, making gene cloning, expression and functional studies convenient as well as time- and labor-efficient

A Plasmid vector encoding functional human keratinocyte growth factor gene in vitro—Functional human KGF gene expression in vitro

In this study, we cloned human KGF (hKGF) genes using RT-PCR techniques and developed a eukaryotic expression plasmid vector capable of directing the expression of functional hKGF. Monolayer culture of human embryo lung fibro-blast (HLF) was used for isolation of total RNA. Then the total RNA was purified and reverse- transcribed into cDNA using an oligo (dT) primer. A full PCR fragment for hKGF was generated and cloned. Restriction digestion and nucleo-tide sequence analysis validated the complete hKGF transcription. The hKGF cDNA fragment was inserted into pEGFP-C2 vector by means of recombinant DNA technology and verified by restriction analysis and sequencing. We have constructed pEGFP-C2-hKGF encoding the green fluorescent protein (GFP). Furthermore, hKGF had the effect on AEC II proliferation. These results suggest that the potential appli-cation of a hKGF plasmid of gene expression should be useful for sustained AEC proliferation, and its in vivo efficacy needs to be validated. Keywords:

Construction of recombinant plasmid and prokaryotic expression in E. Coli and biological activity analysis of human placenta arresten gene

BACKGROUND: The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have been demonstrated to effectively inhibit angiogenesis and consequently the growth of solid cancer. As for the newly identified angiogenesis inhibitor, arresten, some studies have found its high activity on restrainting tumor vessel. This study was to assess the anti-angiogenic activity of arresten. METHODS: The arresten gene was obtained from a healthy puerpera's placenta tissue by the reverse transcriptase-polymerase chain reaction (RT-PCR) method, and molecular cloning to prokaryotic expression plasmid pBV220 by recombination strategy. The prokaryotic expression plasmid pBV220/arr was identified by restriction enzyme digestion and sequenced. The pBV220/arr was transformed into E. coli JM109, DH5α, BL21 and BL21 (DE3) by the CaCl_2 transformation method. The arresten expression level was detected by SDS-PAGE. The expressed product was purlfled, re-naturalized and detected for its biological activity of inhibiting the angiogenesis of chorioallantoic membrane (CAM). RESULTS: The arresten gene was cloned and pBV220/arr was constructed. The arresten expression level of protein was highly increased after pBV220/arr was transformed into E. coli BL21 (DE3). SDS-PAGE showed that the expressed arresten proteins were mainly inclusion bodies and had a molecular weight of 26 kDa. The expressed arresten protein showed evident biological activities. CONCLUSIONS: The successful construction of recombinant plasmid pBV220/arr and the effective expression in E. coil have laid a foundation for further study of its anti-angiogenic function and may pave the way for future antitumor application.

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。

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。

Construction of plant seed-specific expression vectors pSCB and pSCAB and the obtainment of transgenic Brassica napus H165 expressing poly-3-hydroxybutyrate synthetic genes

The seed-specific promoter and transit peptide were amplified and fused to the three genes phbA, phbB and phbC encoding PHB synthetic enzymes, respectively. Seed-specific expression vectors pSCB containing phbC and phbB, and pSCAB containing phbC, phbB and phbA, were constructed by introducing the genes with promoter and peptide into the binary vector pBI101. Transgenic Brassica napus H165 were obtained by Agrobacterium-mediated transformation with these vectors. They were confirmed by PCR, Southern and RT-PCR analyses.

绵羊GSTA2基因克隆、生物信息学分析及真核表达载体构建

为阐明绵羊谷胱甘肽S转移酶α2(Glutathione S-transferase alpha 2,GSTA2)基因功能,利用牛GSTA2基因序列设计引物,提取绵羊皮肤组织总RNA,应用RT-PCR、基因克隆、生物信息学对GSTA2基因进行研究,构建真核表达载体,转染绵羊皮肤成纤维细胞,应用qPCR技术检测GSTA2基因的表达。结果表明,克隆到绵羊GSTA2基因672 bp编码区序列(GenBank登录号:OR440604.1),编码223个氨基酸,分子量为25.39 ku。氨基酸比对和系统进化树分析表明,绵羊与牛的GSTA2相似度最高,达87.39%。GSTA2的二级结构以α-螺旋和无规则卷曲为主。构建的真核表达载体pcDNA3.1-GSTA2转染绵羊皮肤成纤维细胞后,GSTA2基因表达量极显著上调(P <0.001)。说明成功克隆了绵羊新基因GSTA2的CDS序列,并成功构建了其真核表达载体。

鼠源ATP5B基因克隆及其原核和真核表达载体构建

【目的】克隆鼠源ATP5B基因,构建其原核和真核表达载体,为研究ATP5B蛋白功能及其在病毒感染过程中的作用机制提供基础。【方法】克隆鼠源ATP5B基因编码区(CDS),运用ProtParam、ProtScale和SignalP-5.0等对ATP5B蛋白进行生物信息学分析。采用同源重组方法构建原核表达载体pGEX-4T-1-ATP5B-Flag和真核表达载体pcDNA3.0-ATP5B-Flag。以原核表达载体pGEX-4T-1-ATP5B-Flag转化大肠杆菌BL21感受态细胞,利用IPTG进行诱导表达,分析最适诱导温度和IPTG浓度。以真核表达载体pcDNA3.0-ATP5B-Flag转染HEK-293T细胞,通过Western blotting和间接免疫荧光试验(IFA)检测ATP5B蛋白在细胞中的表达及分布情况。【结果】鼠源ATP5B基因CDS长1590 bp,鼠源ATP5B蛋白由529个氨基酸残基构成,分子量为55 kD,理论等电点(pI)为5.21,属于稳定的疏水性蛋白,无跨膜结构域和信号肽,不属于分泌蛋白;鼠源ATP5B蛋白二级结构中α-螺旋占43.67%,无规则卷曲占32.51%,β-转角占9.45%,延伸链占14.37%。原核表达载体pGEX-4T-1-ATP5B-Flag经IPTG诱导后,通过考马斯亮蓝染色和Western blotting在81 kD处成功检测到融合蛋白,且主要以包涵体形式表达,诱导条件以30℃、0.5 mmol/L IPTG的效果更好。以真核表达载体pcDNA3.0-ATP5B-Flag转染HEK-293T细胞后,在55 kD处检测到Flag标签特异性条带,即ATP5B蛋白在HEK-293T细胞中成功表达,IFA检测结果表明该蛋白主要定位在细胞质中。【结论】成功构建鼠源ATP5B基因原核和真核表达载体,原核表达载体表达出的融合蛋白主要以包涵体形式存在,只有少量可溶蛋白,真核表达的ATP5B蛋白主要定位在细胞质中。鼠源ATP5B蛋白是理化性质稳定的疏水性蛋白,无信号肽位点,不属于分泌蛋白。

Cloning, construction of prokaryotic expression vector and expression of Escherichia coli cytosine deaminase gene

Cytosine deaminase gene of Escherichia coli strain H30 was cloned, and its initiation codon of ’GTG’ was mutated to ’ATG’ by PCR. Prokaryotic recombinant expression vector pBV220CD was constructed. Clone with high enzyme activity were selected by detecting their specific activity of cytosine deaminase. 5FC（5FC, 5fluorocytosine） could induce the lethal toxicity to cells containing active CD gene. DNA sequence analysis indicated that there were 16 altered bases and 5 of them resulted in the alteration of amino acids in predicted peptide by comparing DNA sequence of the clone H30CD11 with high enzyme activity with CD gene reported in Gene Bank.

陆地棉小GTP结合蛋白基因GhROP6的克隆及表达初步分析

为探究ROP基因在棉花抵御逆境胁迫中的生物学功能,利用同源克隆的方法获得一个陆地棉GhROP6基因,通过生物信息学方法分析其理化性质、结构及进化关系,利用实时荧光定量PCR(qRTPCR)技术探究GhROP6基因的组织表达特异性及不同逆境胁迫和外源激素处理下的表达模式,构建GhROP6基因的VIGS载体并转化棉花,利用qRT-PCR技术检测其沉默效率。结果显示,GhROP6基因开放阅读框(ORF)为597 bp,编码一个含198个氨基酸的I类ROP蛋白;多重序列比对结果显示,GhROP6符合ROP蛋白结构特征,且与其他物种ROP蛋白高度同源;进化树分析结果显示GhROP6蛋白与拟南芥AtROP6蛋白同源性最高;GhROP6基因在棉花根、茎、真叶及子叶中均有表达,且在真叶中表达量最高;GhROP6基因对干旱、高盐、低温、高温等胁迫和外源脱落酸(ABA)、生长素(IAA)处理均有不同程度的响应,可能在棉花抗逆反应中扮演着重要角色。GhROP6在棉花的叶片和根部均得到有效沉默,表明已获得GhROP6基因沉默植株。本研究为进一步了解GhROP6基因的分子生物学功能奠定了基础。

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.

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.

日本蛇根草OjDFR5基因的克隆与真核表达载体的构建

二氢黄酮醇4-还原酶具有明显的底物特异性,是类黄酮代谢途径中的关键酶,对不同花青素的合成与积累起到决定作用,直接影响植物的颜色性状。日本蛇根草是一种具有较好药用价值的研究材料。通过分子生物学方法克隆获得日本蛇根草中的二氢黄酮醇4-还原酶的编码基因,OjDFR5,并对该基因的序列进行分析,同时将其与真核表达载体pBI121进行连接,获得真核重组表达质粒pBI121-OjDFR5,并将重组质粒转入农杆菌GV3101感受态细胞中。研究结果一方面为该基因的功能解析奠定基础,同时也为探究日本蛇根草类黄酮代谢机制研究提供基因资源。

新麦草IPT基因亚细胞定位、过表达载体构建及鉴定

为了验证IPT基因在新麦草中的功能,为后续新麦草IPT基因进一步功能验证提供试验材料和理论依据。研究以DT型和ST型的新麦草分蘖节为材料,通过RNA-seq分析和qRT-PCR试验验证IPT的相对表达量并进行GO富集分析,采用PCR法克隆IPT基因,构建1300-cYFP-IPT过表达载体,并进行生物信息学分析;通过烟草瞬时转染法和蛋白质印迹法鉴定IPT蛋白的亚细胞定位及表达情况。结果显示,IPT与tRNA二甲基烯丙基转移酶活性相关,在新麦草分蘖中起上调作用;克隆得到新麦草IPT基因全长,并构建了1300-cYFP-IPT过表达载体,其开放阅读框(ORF)为1362 bp;多序列比对与保守结构域分析表明,新麦草IPT蛋白存在PLN02840蛋白保守结构域,与二粒小麦、小麦及硬粒小麦IPT蛋白的亲缘关系较近;蛋白质二级结构预测显示新麦草IPT蛋白二级结构由α-螺旋、延伸链、β-折叠和不规则卷曲组成;分析显示丝氨酸的磷酸化位点最有可能是蛋白发挥功能的潜在磷酸化位点;烟草瞬时转染显示IPT蛋白正常表达,定位于叶绿体中;Western blot结果显示连接载体的目的蛋白IPT正常表达,大小为76.8 kD。研究表明IPT基因在新麦草分蘖过程中上调分蘖数,1300-cYFP-IPT载体可在植株叶绿体中正常表达。

杂色云芝漆酶基因(Lcc1)的克隆及在甲醇毕赤酵母中的表达

以白腐菌杂色云芝Coriolus versicolor RNA为模板,通过RT-PCR获得漆酶Leel基因的cDNA片段。构建了甲醇酵母表达质粒pMETA-Lccl载体,并将其线性化后用电穿孔法导入Pichia methabolica PMAD16,部分阳性克隆的PCR结果表明Lccl基因已经整合到甲醇毕赤酵母染色体上,经摇瓶培养筛选出表达水平较高的酵母工程菌株。

猪胸膜肺炎放线杆菌ApxIVA基因特异片段的克隆和表达

以猪胸膜肺炎放线杆菌的血清7型国内分离株25＿4株基因组DNA为模板,PCR方法扩增apxIVA特异片段,PCR产物经纯化后与载体PMD18＿T进行连接、转化,经酶切及序列分析鉴定后,亚克隆到原核表达载体pGEX＿6P＿1中,构建成重组表达质粒pGEX＿apxIVA,转入到大肠杆菌BL21中,以IPTG进行诱导,进行SDS＿PAGE电泳。结果表明,25＿4株apxIVAgene与基因库中标准7型apxIVAgene同源性达97%,所表达的融合蛋白相对分子量为44kD,与实际预测相符。apxIVA毒素特异片段的成功表达为猪胸膜肺炎放线杆菌病的诊断打下基础。