Direct Agrobacterium tumefaciens-mediated transformation of Hyoscyamus muticus hairy roots using green fluorescent protein

Hyoscyamus muticus hairy root segments were infected with Agrobacterium tumefaciens ASE containing the binary vector pCGN1548 with a green fluorescent protein (GFP) reporter gene under the control of the CaMV 35S promoter. The roots were incubated on callus-inducing medium to generate transformed cells. Transformants were selected on medium containing 50 and 100 mg/L kanamycin and screened by visual inspection for GFP expression. Highly fluorescent cells were incubated on phytohormone-free medium for regeneration of the hairy root phenotype. This infection technique can be applied directly to existing hairy root cultures which have been previously characterized and selected for desirable physiological traits. These studies also indicate that GFP is not toxic to H. muticus plant tissue and that H. muticus hairy roots have minimal autofluorescence which allows for clear observation of GFP.     

A chemically inducible cucumber mosaic virus amplicon system for expression of heterologous proteins in plant tissues

A novel cucumber mosaic virus inducible viral amplicon (CMViva) expression system has been developed that allows for tightly regulated chemically inducible expression of heterologous genes in plant hosts. Transient production of recombinant α₁-antitrypsin (rAAT), a human blood protein, was demonstrated in Nicotiana benthamiana leaves. The highest production levels were obtained by co-infiltrating leaves with Agrobacterium tumefaciens cells containing CMViva carrying the AAT gene and A. tumefaciens cells carrying a binary vector constitutively expressing the gene silencing suppressor p19. Accumulation of up to thirty-fold more rAAT was observed in leaves (24 mg per 100 g leaf tissue) when compared with the expression levels observed using the cauliflower mosaic virus (CaMV) 35S promoter. Significantly, 70% of the rAAT produced using the CMViva expression system was found to be biologically active, a 170-fold increase in functional protein compared with the CaMV 35S expression system.     

Expression Activity of CLCuV Bidirectional Promoter in Agrobacterium tumefaciens

Cotton leaf curl virus (CLCuV) belongs to the subgroup Ⅲ of geminiviruses with single strand DNA genome. Study demonstrated that the bidirectional promoter of CLCuV had activity in Agrobacterium tumefaciens (Smith et Townsend) Conn. This is the first report for the activity of the bidirectional promoter of geminivirus in A. tumefaciens . Results showed that the activity of the complementary sense promoter was stronger than that of virion sense promoter, and was detected 2-fold higher than that of CaMV 35S promoter in A. tumefaciens . Moreover, the promoter 5′ deletion analysis indicated that the mean GUS activity driven by a 287 nucleotides complementary sense promoter fragment (from -287 to the translation initiation site) is 4 times higher than that driven by the whole complementary sense promoter in A. tumefaciens . This result suggested that there might exist negative regulatory elements in this deleted fragment. The function of other cis- elements included in CLCuV complementary sense promoter was also discussed in this paper.     

Assessment of the Effectiveness of a Nuclear-Launched TMV-Based Replicon as a Tool for Foreign Gene Expression in Plants in Comparison to Direct Gene Expression from a Nuclear Promoter

An environmentally safe Tobacco Mosaic Virus (TMV)-based expression replicon was constructed that lacks movement protein (MP) and coat protein (CP), and which expresses the green fluorescent protein (GFP) gene from a full CP subgenomic promoter. The TMV replicon, whose cDNA was positioned between an enhanced Cauliflower Mosaic Virus 35S promoter (CaMV) and a self-cleaving hammerhead ribozyme with a downstream nopaline synthase gene polyadenylation signal [nos-poly(A)], was assessed for its effectiveness to accumulate GFP upon agroinfiltration into plant leaves compared to a control construct in which GFP was directly expressed from the enhanced CaMV 35S promoter. It was determined that individually expressing cells produced ca. 9-fold more GFP from the TMV-based replicon than from the enhanced 35S promoter. In contrast, GFP measurements from total leaf extracts determined that leaves infiltrated with the TMV-based replicon produced ca. 7-fold less GFP than the control construct. These apparently contradictory results can be explained by the low infectivity of the TMV-based replicon as it was found that the number of foci expressing GFP produced in leaves agroinfiltrated with the TMV-based replicon was ca. 66-fold lower than produced by the control.     

Viral and chloroplastic signals essential for initiation and efficiency of translation in Agrobacterium tumefaciens

The construction of high-level protein expression vectors using the CaMV 35S promoter in concert with highly efficient translation initiation signals for Agrobacterium tumefaciens is a relatively less explored field compared to that of Escherichia coli. In the current study, we experimentally investigated the capacity of the CaMV 35S promoter to direct GFP gene expression in A. tumefaciens in the context of different viral and chloroplastic translation initiation signals. GFP expression and concomitant translational efficiency was monitored by confocal microscopy and Western blot analysis. Among all of the constructs, the highest level of translation was observed for the construct containing the phage T7 translation initiation region followed by the chloroplastic Rubisco Large Subunit (rbcL) 58-nucleotide 5′ leader region including its SD-like sequence (GGGAGGG). Replacing the SD-like (GGGAGGG) with non SD-like (TTTATTT) or replacing the remaining 52 nucleotides of rbcL with nonspecific sequence completely abolished translation. In addition, this 58 nucleotide region of rbcL serves as a translational enhancer in plants when located within the 5′ UTR of mRNA corresponding to GFP. Other constructs, including those containing sequences upstream of the coat proteins of Alfalfa Mosaic Virus, or the GAGG sequence of T4 phage or the chloroplastic atpI and/or PsbA 5′ UTR sequence, supported low levels of GFP expression or none at all. From these studies, we propose that we have created high expression vectors in A. tumefaciens and/or plants which contain the CaMV 35S promoter, followed by the translationally strong T7 SD plus RBS translation initiation region or the rbcL 58-nucleotide 5′ leader region upstream of the gene of interest.     

过量表达大叶补血草LgNHX1基因对拟南芥耐盐性的影响

利用植物表达载体pCAMBIA1301和农杆菌GV3101将LgNHX1(全长1 656 bp)基因在拟南芥中过量表达.在含30 mg/L潮霉素的培养基上筛选获得LgNHX1的纯合转化子,并对其进行了分子鉴定和耐盐性分析.结果显示,经PCR和RT-PCR鉴定,野生型植株(对照)没有出现扩增条带,而转基因株系有相应的扩增条带,表明LgNHX1的确已经整合到拟南芥的基因组中,并已正常转录.在不同盐浓度处理下,转基因株系生长情况好于野生型对照;转基因植株地上部分和根的干重、鲜重相对高于野生型对照,但差异没有达到显著水平;当盐浓度达到150-200 mmol/L时,两个特基因株系的Na+含量显著高于野生型,K+含量极显著高于野生型.以上结果表明,过量表达LgNHX1基因可能增强了拟南芥将Na+区隔化至液泡的能力,提高了转基因拟南芥的耐盐能力.     

导入Ghabp1基因对烟草叶细胞发育的影响

将棉花生长素结合蛋白基因cDNA与CaMV35S启动子和NOS终止子融合,构建了一个新的表达载体pGABP1-121,采用农杆菌介导法转化烟草,经过分化、筛选和再生,得到了具有卡那霉素抗性的植株。抗性植株经PCR及Southern杂交检测,证明外源目的基因已经整合到烟草基因组中。扫描电镜观察发现转基因烟草与对照相比叶细胞增大,结果表明,棉花生长素结合蛋白基因的表达影响了烟草叶细胞的发育。     

甘菊DFL::EGFP高效融合蛋白表达载体的构建

增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)是一种优化的突变型GFP,DFL是从甘菊中分离出的LFY基因的同源序列。为了研究DFL基因的功能和表达模式,研究利用小片段克隆法将linker序列插入到EGFP基因5′端启始密码子前面,在pBI121载体的CaMV35S启动子的3′端后面插入一段多克隆位点,成功地构建了pBI-DFL-EGFP表达载体。通过设计特异引物,利用PCR技术扩增得了到拟南芥LFY基因的启动子序列,用粘性末端PCR技术将pBI-DFL-EGFP表达载体中CaMV35S启动子替换成LFY基因启动子,构建成了pLFY-DFL-EGFP表达载体。用含有pBI-DFL-EGFP和pLFY-DFL-EGFP质粒的农杆菌侵染洋葱表皮细胞,在荧光显微镜下分别用蓝光激发,均观测到了荧光。这一结果表明,融合蛋白DFL∷EGFP表达载体构建成功,同时还证明了通过PCR技术克隆到的LFY启动子序列具有启动子功能。     

Tracking of the CaMV-35S promoter performance in GFP transgenic tobacco, with a special emphasis on flowers and reproductive organs, confirmed its predominant activity in vascular tissues

Constitutive promoters are the most common promoters used to drive the expression of various genes in monocots and dicots. Therefore, it is of intense interest to ascertain their expression patterns in various plant species, organs and during their ontogenic development. In this study, the activity of the CaMV 35S promoter in transgenic tobacco plants was assessed. In contrast to other studies, performed rather on the primary transformants (T₀ generation), here, individuals of T₁ and T₂ generations were used. The expression profiles of the CaMV 35S promoter were tracked within various plant organs and tissues using the GFP marker. Special attention was given to floral tissues for which the original data regarding the CaMV 35S expression were obtained. As expected, distinct developmental and organ/tissue specific expression patterns in a plant body were observed. CaMV 35S activity was detected in most of the plant tissues and during different developmental stages. The GFP signal was not visible in dry seeds only, but it became clearly apparent within 24–48 h after sowing onto the medium, what, among other things, enables the discrimination of transgenic and non-transgenic seeds/seedlings. Afterwards, the most pronounced GFP fluorescence intensity was usually visible in various vascular tissues of both, T₁ and T₂ plants, indicating the high promoter activity. A stable manifestation of the promoter was retained in the next T₂ generation without any evident changes or losses of activity, showing the expression stability of the CaMV 35S.     

饲料原料中转基因成分的PCR检测

采用PCR检测方法从饲料的主要原料豆粕、玉米蛋白粉中成功地检出启动子35S (35S-promoter,originated from cauliflower mosaic virus)、终止子NOS (nopaline synthase-terminator,derived from Agrobacterium tumefaciens)、耐除草剂基因EPSPS(5-enolpyruvylshikimate-3-phosphate synthase)和抗虫基因CryIA（b）(delta-endotoxin,evolved from Bacillus thuringiensis subsp.kurstaki)等转基因成分,并通过扩增玉米自身蛋白基因Zein( a protein extracted from corn gluten)及大豆自身基因Lectin(chitin-binding protein)的引物和阴阳性对照、阴阳性质控,避免假阳性、假阴性结果。该方法已在口岸进口饲料原料转基因检测中得到初步应用。 Abstract:Based on the heterogenous genes usually used in transgenic crops,the PCR technique was performed with primers derived from CaMV 35S promoter(35S-promoter,originated from cauliflower mosaic virus),NOS terminator(nopaline synthase-terminator,derived from Agrobacterium tumefaciens),EPSPS(5-enolpyruvylshikimate-3-phosphate synthase) gene,and CryIA(b)(delta-endotoxin,evolved from Bacillus thuringiensis subsp.kurstaki)gene to detect transgenic agents from feed raw materials of soybean dregs and corn gluten meal,respectively.Endogenous corn Zein(a protein extracted from corn gluten) gene,soybean Lectin(chitin-binding protein) gene and negative,positive control were applied for avoiding false results.The method established here has been succeessfully applied in detecting transgenic elements in imported feed raw material.     

Iron accumulation in tobacco plants expressing soyabean ferritin gene

High iron-content transgenic tobacco plants have been produced by transfer via Agrobacterium tumefaciens of soyabean ferritin cDNA under the control of a CaMV 35S promoter. Immunoblot analysis of protein from transgenic tobacco plants suggested mature ferritin subunits are produced by cleavage of transit peptides. The expressed ferritin was observed in the tissues of leaves and stems. The maximal iron content of transformant leaves was approximately 30% higher than leaves from non-transformants. The increased iron content of each transformant was correlated with increases in ferritin content. These results demonstrate the potential of breeding high iron content crops by introduction of the ferritin gene     

Study on the conditions of Cell Transformation in Astragalus sinicus

The conditions of genetic transformation of cells in Astragalus sinicus were studied. The experimental results showed that Agrobacterium tumefaciens strain C58 (pKIW 105), when incubated in medium of low pH and low phosphate concentration in presence of acetosyringone could be induced and activated. When the activated bacteria were used to infect A. sinicus, the GUS gene transient expression in the hypocotyl protoplasts of A. sinicus was immediately and remarkably enhanced. This indicated that the vir gene of A. tumefaciens was activated under the above-mentioned incubation conditions which facilitated T-DNA transfer. In PEG-mediated DNA direct transfer, transient expression of GUS gene was promoted by higher pH and higher Ca2+ concentration of fusion medium. In the same experimental condition, expression of GUS gene under the control of MAS-CaMV 35S chimeric promoter was more effective than that under the control of CaMV 35S promoter, and intensity of GUS gene expression was positively correlated with the amount of foreign plasmid DNA in the range of 10--100 μg. Adventitious shoots were induced from cotyledon and hypocotyls explants treated with Agrobacterium turnefaciens strain PGV 2260 (pBI 121) and were subcultured on MS medium containing 50 mg/L kanamycin to select transformants, and then the transformed shoots were rooted. Stable expression of the foreign genes in the transformed plants was confirmed by assay of neomycin phosphotransferase Ⅱ (NPT Ⅱ ) and β-glucuronidase (GUS) activity.     

Green fluorescent protein as an all-purpose reporter in Petunia

Two critical attributes of a reporter gene are ease of scoring for activity and capacity for expression in all cell types. We have examined a variant of the gene encoding green fluorescent protein,mgfp5, for its ability to meet these criteria in petunia. Under regulation of the Cauliflower Mosaic Virus (CaMV) 35S promoter, GFP was detectable in all vegetative and most floral cell types. Promoters from petuniaadhl andadh2 allowed for production of GFP in those few cell types lacking GFP production from the CaMV 35S promoter, verifying its capacity for expression in all cell types. With the appropriate promoter, GFP fluorescence was thus readily detectable throughout the plant. A potential complication is the green autofluorescence exhibited by some plant tissues. This auto-fluorescence is for the most part distinguishable from that contributed by GFP, but under-scores the need for appropriate controls in GFP-reporter-based experiments. An erratum to this article is available at .