A Comparison of Constitutive Promoters for Expression of Transgenes in Alfalfa (Medicago Sativa)

The activity of constitutive promoters was compared in transgenic alfalfa plants using two marker genes. Three promoters, the 35S promoter from cauliflower mosaic virus (CaMV), the cassava vein mosaic virus (CsVMV) promoter, and the sugarcane bacilliform badnavirus (ScBV) promoter were each fused to the beta-glucuronidase (gusA) gene. The highest GUS enzyme activity was obtained using the CsVMV promoter and all alfalfa cells assayed by in situ staining had high levels of enzyme activity. The 35S promoter was expressed in leaves, roots, and stems at moderate levels, but the promoter was not active in stem pith cells, root cortical cells, or in the symbiotic zones of nodules. The ScBV promoter was active primarily in vascular tissues throughout the plant. In leaves, GUS activity driven by the CsVMV promoter was approximately 24-fold greater than the activity from the 35S promoter and 38-fold greater than the activity from the ScBV promoter. Five promoters, the double 35S promoter, figwort mosaic virus (FMV) promoter, CsVMV promoter, ScBV promoter, and alfalfa small subunit Rubisco (RbcS) promoter were used to control expression of a cDNA from Trichoderma atroviride encoding an endochitinase (ech42). Highest chitinase activity in leaves, roots, and root nodules was obtained in plants containing the CsVMV:ech42 transgene. Plants expressing the endochitinase were challenged with Phoma medicaginis var. medicaginis, the causal agent of spring black stem and leaf spot of alfalfa. Although endochitinase activity in leaves of transgenic plants was 50- to 2650-fold greater than activity in control plants, none of the transgenic plants showed a consistent increase in disease resistance compared to controls. The high constitutive levels of both GUS and endochitinase activity obtained demonstrate that the CsVMV promoter is useful for high-level transgene expression in alfalfa.     

Activity of a chimeric promoter with the doubled CaMV 35S enhancer element in protoplast-derived cells and transgenic plants in maize

A reproducible and efficient transformation system has been developed for maize that is based on direct DNA uptake into embryogenic protoplasts and regeneration of fertile plants from protoplast-derived transgenic callus tissues. Plasmid DNA, containing the -glucuronidase (GUS) gene, under the control of the doubled enhancer element (the –208 to –46 bp upstream fragment) from CaMV 35S promoter, linked to the truncated (up to –389 bp from ATG) promoter of wheat, -amylase gene was introduced into protoplasts from suspension culture of HE/89 genotype. The constructed transformation vectors carried either the neomycin phosphotransferase (NPTII) or phosphinothricin acetyltransferase (PAT) gene as selective marker. The applied DNA uptake protocol has resulted at least in 10–20 resistant calli, or GUS-expressing colonies after treatment of 10⁶ protoplasts. Vital GUS staining of microcalli has made possible the shoot regeneration from the GUS-stained tissues. 80–90% of kanamycin or PPT resistant calli showed GUS activity, and transgenic plants were regenerated from more than 140 clones. Both Southern hybridization and PCR analysis showed the presence of introduced foreign genes in the genomic DNA of the transformants. The chimeric promoter, composed of a tissue specific monocot promoter, and the viral enhancer element specified similar expression pattern in maize plants, as it was determined by the full CaMV 35S promoter in dicot and other monocot plants. The highest GUS specific activity was found in older leaves with progressively less activity in young leaves, stem and root. Histochemical localization of GUS revealed promoter function in leaf epidermis, mesophyll and vascular bundles, in the cortex and vascular cylinder of the root. In roots, the meristematic tip region and vascular tissues stained intensively. Selected transformants were grown up to maturity, and second-generation seedlings with segregation for GUS activity were obtained after outcrossing. The GUS-expressing segregants carried also the NPTII gene as shown by Southern hybridization.     

Cotton α-Globulin Promoter: Isolation and Functional Characterization in Transgenic Cotton,Arabidopsis, and Tobacco

Globulins are the most abundant seed storage proteins in cotton and, therefore, their regulatory sequences could potentially provide a good source of seed-specific promoters. We isolated the putative promoter region of cotton -globulin B gene by gene walking using the primers designed from a cotton staged embryo cDNA clone. PCR amplified fragment of 1108 bp upstream sequences was fused to gusA gene in the binary vector pBI101.3 to create the test construct. This was used to study the expression pattern of the putative promoter region in transgenic cotton, Arabidopsis, and tobacco. Histochemical GUS analysis revealed that the promoter began to express during the torpedo stage of seed development in tobacco and Arabidopsis, and during cotyledon expansion stage in cotton. The activity quickly increased until embryo maturation in all three species. Fluorometric GUS analysis showed that the promoter expression started at 12 and 15 dpa in tobacco and cotton, respectively, and increased through seed maturation. The strength of the promoter expression, as reflected by average GUS activity in the seeds from primary transgenic plants, was vastly different amongst the three species tested. In Arabidopsis, the activity was 16.7% and in tobacco it was less than 1% of the levels detected in cotton seeds. In germinating seedlings of tobacco and Arabidopsis, GUS activity diminished until it was completely absent 10 days post imbibition. In addition, absence of detectable level of GUS expression in stem, leaf, root, pollen, and floral bud of transgenic cotton confirmed that the promoter is highly seed-specific. Analysis of GUS activity at individual seed level in cotton showed a gene dose effect reflecting their homozygous or hemizygous status. Our results show that this promoter is highly tissue-specific and it can be used to control transgene expression in dicot seeds.     

The scutellar vascular bundle-specific promoter of the wheat HD-Zip IV transcription factor shows similar spatial and temporal activity in transgenic wheat, barley and rice

An HD‐Zip IV gene from wheat, TaGL9, was isolated using a Y1H screen of a cDNA library prepared from developing wheat grain. TaGL9 has an amino acid sequence distinct from other reported members of the HD‐Zip IV family. The 3′ untranslated region of TaGL9 was used as a probe to isolate a genomic clone of the TaGL9 homologue from a BAC library prepared from Triticum durum L. cv. Langdon. The full‐length gene containing a 3‐kb‐long promoter region was designated TdGL9H1. Spatial and temporal activity of TdGL9H1 was examined using promoter‐GUS fusion constructs in transgenic wheat, barley and rice plants. Whole‐mount and histochemical GUS staining patterns revealed grain‐specific expression of TdGL9H1. GUS expression was initially observed between 3 and 8 days after pollination (DAP) in embryos at the globular stage and adjacent to the embryo fraction of the endosperm. Expression was strongest in the outer cell layer of the embryo. In developed wheat and barley embryos, strong activity of the promoter was only detected in the main vascular bundle of the scutellum, which is known to be responsible for the uptake of nutrients from the endosperm during germination and the endosperm‐dependent phase of seedling development. Furthermore, this pattern of GUS staining was observed in dry seeds several weeks after harvesting but quickly disappeared during imbibition. The promoter of this gene could be a useful tool for engineering of early seedling vigour and protecting the endosperm to embryo axis pathway from pathogens during grain desiccation and storage.     

Developmental regulation of an acyl carrier protein gene promoter in vegetative and reproductive tissues

The expression of an Arabidopsis acyl carrier protein (ACP) gene promoter has been examined in transgenic tobacco plants by linking it to the reporter gene -glucuronidase (GUS). Fluorometric analysis showed that the ACP gene promoter was most active in developing seeds. Expression was also high in roots, but significantly lower in young leaves and downregulated upon their maturation. Etiolated and light-grown seedlings showed the same level of GUS activity, indicating that this promoter is not tightly regulated by light. Histochemical studies revealed that expression was usually highest in apical/ meristematic zones of vegetative tissues. Young flowers (ca. 1 cm in length) showed GUS staining in nearly all cell types, however, cell-specific patterns emerged in more mature flowers. The ACP gene promoter was active in the stigma and transmitting tissue of the style, as well as in the tapetum of the anther, developing pollen, and ovules. The results provide evidence that this ACP gene is regulated in a complex manner and is responsive to the array of signals which accompany cell differentiation, and a demand for fatty acids and lipids, during organogenesis.     

Characterization of a barley gene coding for an α-amylase inhibitor subunit (CMd protein) and analysis of its promoter in transgenic tobacco plants and in maize kernels by microprojectile bombardment

A gene coding for a barley CMd protein was isolated from a genomic library using a cDNA probe encoding the wheat CM3 protein. Promoter sequence analysis reveals motifs found in genes specifically expressed in endosperm and aleurone cells, as well as TATA and other putative functional boxes. 720 bp of the Hv85.1 CMd protein gene promoter, when fused to a gus coding region, were unable to direct GUS activity in the seeds of transgenic tobacco plants. In contrast, the same construction delivered into immature maize kernels by microprojectile bombardment was able to direct expression of GUS in the outermost cell layers of maize endosperm in both a tissue-specific and a developmentally determined manner.     

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the -glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.     

Stability of β-glucuronidase gene expression in transgenic Tricyrtis hirta plants after two years of cultivation

Transgenic plants of Tricyrtis hirta carrying the intron-containing β-glucuronidase (GUS) gene under the control of the CaMV35S promoter have been cultivated for two years. Four independent transgenic plants produced flowers 1–2 years after acclimatization, and all of them contained one copy of the transgene as indicated by inverse polymerase chain reaction (PCR) analysis. All the four transgenic plants showed stable expression of the gus gene in leaves, stems, roots, tepals, stamens and pistils as indicated by histochemical and fluorometric GUS assays, although differences in the GUS activity were observed among different organs of each transgenic plant. No apparent gus gene silencing was observed in transgenic T. hirta plants even after two years of cultivation.     

Effects of antibiotics on the elimination of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> from loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis>) zygotic embryo explants and on transgenic plant regeneration

Three antibiotics were evaluated for their effects on the elimination of Agrobacterium tumefaciens during the genetic transformation of loblolly pine ( Pinus taeda L.) using mature zygotic embryos as targets. Agrobacterium tumefaciens strains, EHA105, GV3101, and LBA 4404, all harbouring the plasmid pCAMBIA1301, which carries the selectable marker gene, hygromycin phosphotransferase ( hpt) controlled by the cauliflower mosaic virus 35S promoter and terminator, and the uidA reporter gene (GUS) driven by the cauliflower mosaic virus 35S promoter and the terminator of nopaline synthase gene, were used in this study. Exposure to 350 mg l^-1 carbenicillin, claforan, and timentin respectively for up to 6 weeks did not eliminate the Agrobacterium, while antibiotics at 500 mg l^-1 eradicated them from the co-cultivated zygotic embryos. All three antibiotics increased callus growth and shoot regeneration at 350 and 500 mg l^-1 each, but reduced callus growth and shoot regeneration at 650 mg l^-1 when compared with controls. Putative transgenic calli were selected for continued proliferation and differentiation on 4.5 mg l^-1 hygromycin-containing medium. Transformed calli and transgenic plants produced on a selection medium containing 4.5 mg l^-1 hygromycin were confirmed by GUS histochemical assays, by polymerase chain reaction (PCR), and by Southern blot analysis. These results are useful for future studies on optimizing genetic transformation procedures in loblolly pine.     

Xylem-specific expression of a GRP1.8 promoter::4CL gene construct in transgenic tobacco

Lignin is a complex aromatic polymer of vascular plants that provides mechanical strength to the stem and protects cellulose fibres from chemical and biological degradation. 4-Coumarate:CoA ligases (EC 6.2.1.12) are key enzymes for the biosynthetic pathway of monolignols which is an important complex aromatic polymer for lignin biosynthesis and tree growth. Recently, 4-coumarate:CoA ligase has been used as exogenous gene in transgenic plants to genetically modify the lignin biosynthesis pathway. Since most lignin is produced in the vascular cells, a tissue-specific-expressed promoter in the vascular cell would be important and useful to change and modify the content of lignin. Here we report the existence of a promoter of GRP1.8 (the glycine-rich protein 1.8) in Sopho japonica L. (GenBank accession number AF250149) and studies on its function in transgenic tobacco. The promoter activity was analyzed in transgenic tobacco plants by histochemical staining of GUS gene expression driven by a 613-bp sjGRP1.8p promoter sequence. In sjGRP1.8p-GUS transgenic plants, intense GUS staining was detected in the xylem of the stem. To further investigate the regulation of the tissue-specific expression of the 4CL1 gene, we analyzed the activity of the 4CL1 gene which is sense orientated with the sjGRP1.8p promoter in transgenic tobacco. The Pto4CL1 gene was expressed in the stem of transgenic tobacco. The activity of the 4CL1 enzyme was increased 1–2-fold in the stem but not increased in the leaves of transgenic tobacco. In comparison with the control plants, the content of lignin was increased 25% in the stem but there was no increase in the leaves of transgenic tobacco.     

Agrobacterium-mediated production of transgenic rice plants expressing a chimeric α-amylase promoter/β-glucuronidase gene

We have successfully transferred and expressed a reporter gene driven by an -amylase promoter in a japonica type of rice (Oryza sativa L. cv. Tainung 62) using the Agrobacterium-mediated gene transfer system. Immature rice embryos (10–12 days after anthesis) were infected with an Agrobacterium strain carrying a plasmid containing chimeric genes of -glucuronidase (uidA) and neomycin phosphotransferase (nptII). Co-incubation of potato suspension culture (PSC) with the Agrobacterium inoculum significantly improved the transformation efficiency of rice. The uidA and nptII genes, which are under the control of promoters of a rice -amylase gene (Amy8) and Agrobacterium nopaline synthase gene (nos), respectively, were both expressed in G418-resistant calli and transgenic plants. Integration of foreign genes into the genomes of transgenic plants was confirmed by Southern blot analysis. Histochemical localization of GUS activity in one transgenic plant (R0) revealed that the rice -amylase promoter functions in all cell types of the mature leaves, stems, sheaths and roots, but not in the very young leaves. This transgenic plant grew more slowly and produced less seeds than the wild-type plant, but its R1 and R2 progenies grew normally and produced as much seeds as the wild-type plant. Inheritance of foreign genes to the progenies was also confirmed by Southern blot analysis. These data demonstrate successful gene transfer and sexual inheritance of the chimeric genes.     

Spatial and temporal expression patterns directed by theAgrobacterium tumefaciens T-DNA gene 5 promoter during somatic embryogenesis in carrot

We have analysed the patterns of expression of a gene encoding -glucuronidase (GUS) fused to the promoter of theAgrobacterium tumefaciens T-DNA gene 5 during embryogenesis in carrot,Daucus carota L. Gene expression was monitored by a histochemical assay of -glucuronidase activity. The gene 5 promoter, although of bacterial origin, conferred expression upon the marker gene in all stages of embryo development. The patterns of expression however, differed between embryos in different stages of development. In the globular stage expression was confined to the basal part of the embryo, suggesting that the promoter is sensitive to regulatory functions active in the primary establishment of polarity in the radially symmetric globular embryo. In the heart and torpedo stages of development GUS expression was high in the entire embryonic axis, but not in the cotyledons. During germination expression was reduced in the elongating hypocotyl and radicle, and high levels of expression were detected only in the shoot and root apices. Among the transformed cell lines analysed, one was found that showed an aberrant pattern of GUS expression during embryogenesis, in that expression in the upper part of the embryo was undetectable, and expression was restricted to the root apex in later stages of development. This difference in organ specificity of expression is likely due to a large deletion of the promoter.     

大麦与油菜种皮特异启动子表达模式的比较

启动子位于转录起始位点上游并能特异性地结合RNA聚合酶,其作为调控序列驱动外源基因在异源植物中表达,从而实现转基因的高效性,具有时空表达特异性的启动子对获得有效转基因植物及产物具有重要意义。为了解种皮特异启动子的表达模式,该研究基于前期报道的序列,通过同源克隆的方法分别从大麦和油菜中克隆获得Gerb和Bntt两个种皮特异性启动子,并对其进行生物信息学分析,构建了Gerb::GUS和Bntt::GUS植物表达载体并转化拟南芥,通过组织化学染色观察了GUS的表达情况。结果表明:两种启动子序列中都含有多拷贝种皮特异表达启动子元件以及多种胁迫诱导响应元件;转基因拟南芥幼苗期,大麦Gerb种皮特异启动子驱动GUS全株表达且子叶和下胚轴较真叶和根中表达量高;油菜Bntt种皮特异启动子表达较弱;成株期,Gerb在不同组织(叶片、茎、花序和角果)中均有表达,未显示组织特异性;Bntt仅在叶片及角果维管束中有微弱表达。在各种非生物胁迫下,Gerb表达模式未发生显著变化,而Bntt仅在盐胁迫下显示很强的角果和种子特异性表达,其他胁迫未见明显表达。以上结果显示,大麦种皮特异性启动子Gerb和油菜种皮特异性启动子Bntt在时间和空间表达模式上存在差异,这对今后选择种皮特异启动子具有参考作用,但其具体机制仍需进一步研究验证。     

Molecular and biochemical characterization of three aromatic polyketide synthase genes from Rubus idaeus

To play an essential role in C₄ photosynthesis, the maize C₄ phosphoenolpyruvate carboxylase gene (PPCZm1) acquired many new expression features, such as leaf specificity, mesophyll specificity, light inducibility and high activity, that distinguish the unique C₄ PPC from numerous non-C₄ PPC genes in maize. We present here the first investigation of the developmental, cell-specific, light and metabolic regulation of the homologous C₄ PPCZm1 promoter in stable transgenic maize plants. We demonstrate that the 1.7 kb of the 5-flanking region of the PPCZm1 gene is sufficient to direct the C₄-specific expression patterns of -glucuronidase (GUS) activity, as a reporter, in stable transformed maize plants. In light-grown shoots, GUS expression was strongest in all developing and mature mesophyll cells in the leaf, collar and sheath. GUS activity was also detected in mesophyll cells in the outer husks of ear shoots and in the outer glumes of staminate spikelets. We did not observe histological localization of GUS activity in light- or dark-grown callus, roots, silk, developing or mature kernels, the shoot apex, prop roots, or pollen. In addition, we used the stable expressing transformants to conduct and quantify physiological induction studies. Our results indicate that the expression of the C₄ PPCZm1-GUS fusion gene is mesophyll-specific and influenced by development, light, glucose, acetate and chloroplast biogenesis in transgenic maize plants. These studies suggest that the adoption of DNA regulatory elements for C₄-specific gene expression is a crucial step in C₄ gene evolution.     

Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana

Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic -glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F₁ and F₂ progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F₁ progeny and most of the F₂ progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F₂ individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F₁ generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.     

Visual characterization of recombination at FRT-gusA loci in transgenic tobacco mediated by constitutive expression of the native FLP recombinase

FLP/FRT-mediated site-specific recombination was studied with a recombination-reporter gene system which allows visualization of -glucuronidase (GUS) expression after site-specific excisional activation of a silent gusA gene. This system was used for characterization of the functional activity of the Saccharomyces cerevisiae native FLP recombinase driven by the cauliflower mosaic virus (CaMV) 35s promoter [linked to the tobacco mosaic virus (TMV) omega translational leader] in mediating site-specific recombination of chromosomal FRT sites in tobacco FLP x FRT-reporter hybrids. Six hybrids were generated from crosses of lines containing either a stably integrated recombination-reporter or a FLP-expression construct. The activated gusA phenotype was specific to hybrid progenies and was not observed in either parental plants or their selfed progenies. Recombination efficiency in whole seedlings was estimated by the percent of radioactivity on a Southern blot which was incorporated into the recombined DNA product. Estimated efficiency mean values for the six crosses ranged from 5.2 to 52.0%. Histochemical analysis in hybrid plants visualized GUS activity with variable chimeric patterns and intensities. Recombination efficiency and GUS expression varied both among and within crosses, while higher recombination efficiency coincided with larger and more intense patterns of GUS activity. These data suggest that recombination is induced randomly during somatic developmental stages and that the pattern and intensity generated in a given plant are affected by factors imposing varibility not only between but also within crosses. Additionally, while recombination in a population of FLP/FRT hybrids may occur in all plants, recombination efficiency may still be low in any given plant. The activity of the native, as compared to a modified, FLP (Kilby et al. 1995) in the activation of transgenic traits in tobacco is discussed.