Agrobacterium tumefaciens-mediated sorghum transformation using a mannose selection system

A dual-marker plasmid containing the selectable marker gene, manA, and the reporter gene, sgfp, was used to transform immature sorghum embryos by employing an Agrobacterium-mediated system. Both genes were under the control of the ubi1 promoter in a binary vector pPZP201. The Escherichia coli phosphomannose isomerase (PMI) gene, pmi, was used as the selectable marker gene and mannose was used as the selective agent. The sgfp gene encoding green fluorescence protein (GFP) was the reporter gene and served as a visual screening marker. A total of 167 transgenic plants were obtained from nine different embryogenic callus lines grown on a selection medium containing 1%-2% mannose. Embryoids and shoots regenerated via embryogenesis, that showed strong GFP fluorescence, were selected from two sorghum genotypes: C401, an inbred line, and Pioneer 8505, a commercial hybrid. The GFP accumulation in transgenic plants was observed with a dissecting stereomicroscope. The integration and expression of the manA gene was confirmed by Southern blot and Western blot analyses, and the feasibility of manA selection was demonstrated by the chlorophenol red (CPR) assay. Our results indicated that transgenes segregated in the Mendelian fashion in the T1 generation. The conversion of mannose to a metabolizable fructose carbon source is beneficial to plants. In addition, except in soybean and a few legumes, no endogenous PMI activity has been detected in plant species, indicating that PMI is useful in the transformation of sorghum. In addition, PMI has no sequence homology to known allergens. Optimization of this selection system for sorghum transformation provides an efficient way to produce transgenic plants without using antibiotic or herbicidal agents as selectable markers, and our results showed that the transformation efficiency reached 2.88% for Pioneer 8505 and 3.30% for C401, both values higher than in previously published reports.     

Optimization of sorghum transformation parameters using genes for green fluorescent protein and beta-glucuronidase as visual markers

Early and reliable detection of plant transformation events is essential for establishing efficient transformation protocols. We have compared the effectiveness of using the gene encoding a green fluorescent protein (GFP) and a beta-glucuronidase (gus) as reporter genes for early detection of transgene expression in explants subjected to biolistic bombardment and Agrobacterium-mediated transformation. The results indicate that gfp gene is superior to gus gene in following transgene expression in transiently transformed materials in both methods of transformation. Using GFP as the screenable marker, we have optimized sorghum transformation with respect to the conditions for transformation, type of explants, promoters, and inbreds. These optimized conditions have been used to obtain stably transformed explants for subsequent regeneration.     

Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans

Green fluorescent protein (GFP) is a useful reporter to follow the in vivo behaviour of proteins, but the wild-type gfp gene does not function in many organisms, including many plants and filamentous fungi. We show that codon-modified forms of gfp , produced for use in plants, function effectively in Aspergillus nidulans both as gene expression reporters and as vital reporters for protein location. To demonstrate the use of these modified gfp s as reporter genes we have used fluorescence to follow ethanol-induced GFP expression from the alcA promoter. Translational fusions with the modified gfp were used to follow protein location in living cells; plant ER-retention signals targeted GFP to the endoplasmic reticulum, whereas fusion to the GAL4 DNA-binding domain targeted it to the nucleus. Nuclear-targeted GFP allowed real-time observation of nuclear movement and division. These modified gfp genes should provide useful markers to follow gene expression, organelle behaviour and protein trafficking in real time.     

Stable chloroplast transformation of immature scutella and inflorescences in wheat (Triticum aestivum L.)

Chloroplast transformation in wheat was achieved by bombardment of scutella from immature embryos and immature inflorescences, respectively. A wheat chloroplast site-specific expression vector, pBAGNRK, was constructed by placing an expression cassette containing neomycin phosphotransferase II (nptII) and green fluorescent protein (gfp) as selection and reporter genes, respectively, in the intergenic spacer between atpB and rbcL of wheat chloroplast genome. Integration of gfp gene in the plastome was identified by polymerase chain reaction (PCR) analysis and Southern blotting using gfp gene as a probe. Expression of GFP protein was examined by western blot. Three positive transformants were obtained and the Southern blot of partial fragment of atpB and rbcL (targeting site) probes verified that one of them was homoplasmic. Stable expression of GFP fluorescence was confirmed by confocal microscopy in the leaf tissues from T(1) progeny seedlings. PCR analysis of gfp gene also confirmed the inheritance of transgene in the T(1) progeny. These results strengthen the feasibility of wheat chloroplast transformation and also give a novel method for the introduction of important agronomic traits in wheat through chloroplast transformation.     

Inheritance and expression of introduced DNA in transgenic onion plants (Allium cepa)

Transgenic onion plants (Allium cepa) containing the Cauliflower mosaic virus 35s promoter (CaMV35s) and gfp gene construct encoding the visual green fluorescent reporter protein from pBINm gfp ER and the CaMV35s‐bar gene construct encoding resistance to the herbicide phosphinothricin from pCAMBlA3301 were produced by Agrobacterium‐mediated transformation. These plants weregrown to maturity and selfed in order to determine the expression and inheritance of the transgenes. CaMV35s regulation in onion, as observed by GFP expression, was essentially constitutive, and profiles of regulation were typical of those observed in dicotyledonous plants. Inhibition of CaMV35s regulated gene expression was only observed in one transformant. Both the expression of GFP and tolerance to phosphinothricin appeared to be inherited in a Mendelian fashion. Levels of expression in F1 offspring varied, presumably due to environmental and genetic factors. However, it appeared that copy number did strongly influence GFP protein production and expression. In the majority of plants there were no obvious detrimental phenotypic effects caused by the transgene, the integration event, or Somaclonal variation due to the need to perform tissue culture.     

Quantitative analysis of gene expression with an improved green fluorescent protein. p6.

The fast and easy in vivo detection predestines the green fluorescent protein (GFP) for its use as a reporter to quantify promoter activities. We have increased the sensitivity of GFP detection 320-fold compared to the wild-type by constructing gfp+, which contains mutations improving the folding efficiency and the fluorescence yield of GFP+. Twelve expression levels were measured using fusions of the gfp+ and lacZ genes with the tetA promoter in Escherichia coli. The agreement of GFP+ fluorescence with beta-galactosidase activities was excellent, demonstrating that the gfp+ gene can be used to accurately quantify gene expression in vivo. However, expression of the gfp+ gene from the stronger hsp60 promoter revealed that high cellular concentrations of GFP+ caused an inner filter effect reducing the fluorescence by 50%, thus underestimating promoter activity. This effect is probably due to the higher absorbance of cells containing GFP+. Thus promoters with activities differing by about two orders of magnitude can be correctly quantified using the gfp+ gene. Possibilities of using GFP variants beyond this range are discussed.     

用绿色荧光蛋白监测转基因植物中选择标记基因的消除

绿色荧光蛋白(GFP)可直接进行活体观察,它的这个优点可被用于监测转基因植物中选择标记基因的消除。为此,构建了植物表达载体pGNG,将绿色荧光蛋白基因(gfp)和卡那霉素抗性基因表达盒(NosP-nptll-NosT)一起克隆在两个同向的lox位点间,在第一个lox位点上游置有CaMV 35S启动子以驱动GFP表达,第二个lox位点下游置有不含启动子的大肠杆菌β-葡萄糖醛酸酶(GUS)基因。首先在含卡那霉素(Kan)的培养基上筛选出转pGNG的烟草,借助绿色荧光可容易地检出表达GFP的转化体。然后用另一转化载体pCambia1300Cre二次转化表达GFP的转基因植物,利用另一选择标记基因潮霉素抗性基因(hpt)进行筛选,在获得的再生植株中,Cre重组酶的表达消除了转化体中两lox位点间的gfp和nptll。实验结果表明可借助GFP荧光的消失,快速选出nptII被消除的二次转化体,同时GUS(作为目的蛋白) 在CaMV 35S启动子驱动下获得表达。最后利用后代的分离将hpt和cre除去。     

Site-specific recombination-based genetic system for reporting transient or low-level gene expression

We report here the construction, characterization, and application of a plasmid-based genetic system that reports the expression of a target promoter by effecting an irreversible, heritable change in a bacterial cell. This system confers strong repression of the reporter gene gfp in the absence of target promoter expression and utilizes the site-specific recombination machinery of bacteriophage P22 to trigger high-level reporter gene expression in the original cell and its progeny after target gene induction. We demonstrate the effectiveness of this genetic system by tailoring it to indicate the availability of arabinose to the biological control agent Enterobacter cloacae JL1157 in culture and in the barley rhizosphere. The presence of bioavailable arabinose triggered the production of P22 excisionase and integrase from the reporter plasmid pAraLHB in JL1157, and this led to excision of the cI repressor gene, which is flanked by att sites, and the subsequent irreversible expression of gfp in the original cell and in its progeny. In culture, nearly 100% of an E. cloacae JL1157(pAraLHB) population expressed gfp after exposure to 6.5 to 65 microM arabinose for 3 h. We used this biosensor to demonstrate that arabinose was released from the seeds of several legumes and grass species during germination and from roots of barley seedlings grown hydroponically or in soil. When introduced into microcosms containing barley, the biosensor permitted the localization of arabinose along the roots. Arabinose was present near the root-seed junction and on the seminal roots but was not detected at the root tips. This recombination-based reporter system should be useful for monitoring bacterial exposure to transient or low levels of specific molecules directly in the environment.     

Transient expression of green fluorescent protein in various plastid types following microprojectile bombardment

The green fluorescent protein gene ( gfp ) is a widely used reporter in both animals and plants. Fusions between the plastid rrn promoter or the Escherichia coli trc promoter and the gfp coding region have been delivered to chloroplasts using gold or tungsten microprojectiles, and fluorescence from GFP was visible in individual tobacco chloroplasts and in the abnormally large chloroplasts of the arc 6 mutant of Arabidopsis thaliana 2–4 days after bombardment. The fusion of the gfp coding region to the bacterial trc promoter demonstrated that a bacterial promoter is active in chloroplasts in vivo . GFP was also detectable in amyloplasts of potato tubers and in chromoplasts of marigold petals, carrot roots and pepper fruits 4 days after bombardment. This demonstrates that GFP can be used as a reporter for transient gene expression in chloroplasts and in non-photosynthetic plastids in a range of higher plants.     

Direct detection and quantification of horizontal gene transfer by using flow cytometry and gfp as a reporter gene

A new cultivation-independent method for studying conjugal gene transfer between bacteria was evaluated. The method was based on direct detection and enumeration of donor and transconjugant bacterial cells by flow cytometry. Specific detection of transconjugants was obtained by using a conjugative plasmid tagged with a reporter gene (gfp) encoding green fluorescent protein. A chromosomal encoded repressor (lacI(ql)) repressed expression of GFP in the donor bacteria. Enumeration of the donor cells was performed after induction of GFP expression by the addition of inducer isopropyl-thio-beta-D-galactoside (IPTG). The method presented here provided simple and precise quantification of horizontal gene transfer between both Escherichia coli and Pseudomonas putida strains.     

Mini-transposon Tn5gfp constructs for differential tagging of microorganisms

Recently thegfp (green fluorescent protein) gene from the jellyfishAequoria victoria has been widely used as a reporter gene. In this study mini-transposons, named as mini-Tn5gfp, were constructed by subcloning thegfp gene into a transposon Tn5. To improve the expression level of thegfp gene, tandom array ofgfp gene was obtained. The constructs were successfully used in tagging target microorganisms by transposition. The level of GFP expression was found to be closely correlated with the copy number of the gfp transposed. These constructs will facillitate not only efficient tagging of whole organism but also genetic marking of target genes by transposition.     

In Planta visual monitoring of green fluorescent protein in transgenic rice plants

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a widely used reporter that can be directly visualized in the living cells in both animals and plants. We inserted a synthetic gene (sgfp) encoding a modified form of the GFP into expression vector, Act1-sgfp for the direct expression of GFP which is easily detectable in rice plants. Green fluorescence emitted from GFP could be visualized in calli, dry seeds, roots and seedlings with green shoots of transgenic rice plants. In our visualization system with a charge-coupled device camera, band-pass filters and a light source, the presence of red chlorophyll autofluorescence from chloroplasts did not alter the green fluorescence of GFP. These results demonstrate that GFP could be used as a non-destructive visual selection marker for examining gene expression in transformed calli, dry seeds and young plants.     

绿色荧光蛋白基因作为报告基因在水稻基因转化中的应用研究

本研究中 ,构建了含有编码绿色荧光蛋白的改进型基因质粒pJPM5。用基因枪法分别把pJPM5和另一带有绿色荧光蛋白基因的质粒pSBG70 0转入水稻TNG6 7愈伤组织。用South ern杂交法证实了转基因的存在 ,而且表明多数转基因植株含有 1到 8个拷贝的转基因。取 2个月的转基因植株上的叶片用于分析绿色荧光蛋白基因表达。用SLM - 80 0 0荧光分析仪定量测定绿色荧光蛋白。多数转基因植株具有很高的绿色荧光蛋白信号。虽然水稻植株有少量自发荧光 ,但是绿色荧光蛋白基因表达出的绿色荧光蛋白信号比植株的自发荧光强得多 ,其测定不会受自发荧光的太大影响。在荧光显微镜下观察到了绿色荧光蛋白基因的表达。借助观察分析绿色荧光蛋白基因的瞬时表达 ,本研究还发现基因枪法转化中 ,如果两枪的气压为90 0psi& 135 0psi,比两枪的气压都为 90 0psi或者 135 0psi更好 ,因其能使质粒进入更多的细胞。研究结果表明 ,绿色荧光蛋白基因可以作为水稻 (甚至小麦、玉米 )转基因研究中的报告基因。研究还显示 ,MAR序列能明显增强绿色荧光蛋白基因的表达能力 (这一结果在另文讨论 ) .     

A green fluorescent protein-based whole-cell bioreporter for the detection of phenylacetic acid

Phenylacetic acid (PAA) is produced by many bacteria as an antifungal agent and also appears to be an environmentally toxic chemical. The object of this study was to detect PAA using Pseudomonas putida harboring a reporter plasmid that has a PAA-inducible promoter fused to a green fluorescent protein (GFP) gene. Pseudomonas putida KT2440 was used to construct a green fluorescent protein-based reporter fusion using the paaA promoter region to detect the presence of PAA. The reporter strain exhibited a high level of gfp expression in minimal medium containing PAA; however, the level of GFP expression diminished when glucose was added to the medium, whereas other carbon sources, such as succinate and pyruvate, showed no catabolic repression. Interestingly, overexpression of a paaF gene encoding PAACoA ligase minimized catabolic repression. The reporter strain could also successfully detect PAA produced by other PAA-producing bacteria. This GFP-based bioreporter provides a useful tool for detecting bacteria producing PAA.     

Endosperm-specific expression of green fluorescent protein driven by the hordein promoter is stably inherited in transgenic barley (Hordeum vulgare) plants

The expression of green fluorescent protein (GFP) and its inheritance were studied in transgenic barley (Hordeum vulgare L.) plants transformed with a synthetic green fluorescent protein gene [sgfp(S65T)] driven by either a rice actin promoter or a barley endosperm-specific d-hordein promoter. The gene encoding phosphinothricin acetyltransferase (bar), driven by the maize ubiquitin promoter and intron, was used as a selectable marker to identify transgenic tissues. Strong GFP expression driven by the rice actin promoter was observed in callus cells and in a variety of tissues of T0 plants transformed with the sgfp(S65T)-containing construct. GFP expression, driven by the rice actin promoter, was observed in 14 out of 17 independent regenerable transgenic callus lines; however, expression was gradually lost in T0 and later generation progeny of diploid lines. Stable GFP expression was observed in T2 progeny from only 6 out of the 14 (43%) independent GFP-expressing callus lines. Four of the 8 lines not expressing GFP in T2 progeny, lost GFP expression during T0 plant regeneration from calli; one lost GFP expression in the transition from the T0 to T1 generations and three lines were sterile. Similarly, expression of bar driven by the maize ubiquitin promoter was lost in T1 progeny; only 21 out of 26 (81%) independent lines were Basta-resistant. In contrast to actin-driven expression, GFP expression driven by the d-hordein promoter exhibited endosperm-specificity. All seven lines transformed with d-hordein-driven GFP (100%) expressed GFP in the T1 and T2 generations, regardless of ploidy levels, and expression segregated in a Mendelian fashion. We conclude that the sgfp(S65T) gene was successfully transformed into barley and that GFP expression driven by the d-hordein promoter was more stable in its inheritance pattern in T1 and T2 progeny than that driven by the rice actin promoter or the bar gene driven by the maize ubiquitin promoter.     

phiC31 Integrase-Mediated Site-Specific Recombination in Barley

The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F₁, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.