CaMV 35S promoter directs β-glucuronidase expression in Ganoderma lucidum and Pleurotus citrinopileatus

The cauliflower mosaic virus (CaMV) 35S promoter has been most commonly used in plant transformation studies, but its activity in mushrooms has not been reported. p301-b is a binary vector containing a bialaphos resistance gene driven by the promoter of Lentinus edodes glyceraldehyde-3-phosphate dehydrogenase (GPD) gene. CaMV 35S-GUS was inserted into p301-b, and the resulting construct p301-bG was transformed to protoplasts of Ganoderma lucidum and basidiospores of Pleurotus citrinopileatus. GUS activity was observed in the transformants, indicating that CaMV 35S promoter can direct expression of exogenous gene in the mushrooms. This is the first report on the application of CaMV 35S promoter in genetic modification of mushrooms.     

In vitro mycorrhization of the rubber tree Hevea brasiliensis Müll Arg

In vitro cultivation systems of arbuscular mycorrhizal fungi are useful tools to study the interaction between plants and their fungal symbiont, and also to develop new biotechnologies. Plantlets of the latex-producing species Hevea brasiliensis clone PB 260 were grown in a dense extraradical mycelium network of the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833 developed from a mycelium donor plant (Medicago truncatula A17). The factors indole-3-butyric acid (IBA), 2-morpholineoethanesulfonic acid monohydrate (MES) buffer, and carbon dioxide (CO₂) were tested on root development and colonization by the fungus. No colonization was observed in the presence of plantlets pre-treated with IBA. The highest levels of root colonization were obtained when plantlets were mycorrhized under a high CO₂ concentration (1,000 μmol?mol^?1) with MES (10 mM) added to the growth medium. Widespread root colonization (with presence of arbuscules, intraradical mycelium, and spores/vesicles) was predominantly observed in newly produced roots. Therefore, it appears essential to improve root initiation and growth for improving in vitro mycorrhization of H. brasiliensis. We demonstrated the potential of the “mycelium donor plant” in vitro culture system to produce colonized H. brasiliensis plantlets before their transfer to ex vitro conditions.     

Organ-specific expression of β-glucuronidase activity driven by the Arabidopsis heat-shock promoter in heat-stressed transgenic Nicotiana plumbaginifolia

 The expression of the Arabidopsis heat-shock protein (HSP) 18.2 promoter β-d-glucuronidase (GUS) chimera gene was studied in various organs of the transgenic Nicotiana plumbaginifolia during the recovery phase at normal temperatures (20–22  °C) following heat-shock (HS) treatment. The optimum HS temperature for GUS activity in the anthers, petals and capsules was 42  °C, but in immature seeds and the placentas of capsules it was 36  °C and 39  °C, respectively. No apparent GUS activity was observed in any organs except for dry seeds after HS at 45  °C, although the activity in dry seeds was apparent even after HS at 48  °C. After HS at 42  °C, GUS activity in the flower tissues was the highest before anthesis and declined thereafter. Received: 13 January 1998 / Revision received: 25 January 1999 / Accepted: 3 March 1999     

The biosynthesis of rubber from β-hydroxy-β-methylglutaryl-coenzyme A in Hevea brasiliensis latex

1. beta-Hydroxy-beta-methyl[3-(14)C]glutaryl-CoA is efficiently incorporated into rubber on incubation with Hevea brasiliensis latex, and the incorporation is diminished in the presence of unlabelled mevalonate. beta-Hydroxy-beta-methylglutaric acid is not utilized for rubber synthesis, but inhibits the formation of rubber from beta-hydroxy-beta-methylglutaryl-CoA. 2. The incorporation of beta-hydroxy-beta-methylglutaryl-CoA into rubber is stimulated equally by NADP(+) and NADPH and less so by NAD(+) and NADH. ATP is slightly stimulatory and CoA is inhibitory. 3. beta-Hydroxy-beta-methylglutaryl-CoA reductase is concentrated in the sediment (bottom fraction) formed by centrifuging latex at low speed and the enzyme is unstable in the absence of cysteine or GSH. The formation of NADPH takes place in the latex serum. 4. There is a marked seasonal variation in the extent of beta-hydroxy-beta-methylglutaryl-CoA incorporation into rubber in latex, but mevalonate incorporation is relatively constant. This observation, together with the finding that beta-hydroxy-beta-methylglutaryl-CoA reduction is the rate-limiting step in the formation of rubber from beta-hydroxy-beta-methylglutaryl-CoA, suggests that the conversion of beta-hydroxy-beta-methylglutaryl-CoA into mevalonate is of importance in the regulation of rubber synthesis. 5. Evidence suggesting that beta-hydroxy-beta-methylglutaryl-CoA lyase is present in H. brasiliensis latex has been obtained.     

The sucrose synthase-1 promoter from Citrus sinensis directs expression of the β-glucuronidase reporter gene in phloem tissue and in response to wounding in transgenic plants

Interest in phloem-specific promoters for the engineering of transgenic plants has been increasing in recent years. In this study we isolated two similar, but distinct, alleles of the Citrus sinensis sucrose synthase-1 promoter (CsSUS1p) and inserted them upstream of the β-glucuronidase (GUS) gene to test their ability to drive expression in the phloem of transgenic Arabidopsis thaliana and Nicotiana tabacum. Although both promoter variants were capable of conferring localized GUS expression in the phloem, the CsSUS1p-2 allele also generated a significant level of expression in non-target tissues. Unexpectedly, GUS expression was also instigated in a minority of CsSUS1p::GUS lines in response to wounding in the leaves of transgenic Arabidopsis. Deletion analysis of the CsSUS1p suggested that a fragment comprising nucleotides −410 to −268 relative to the translational start site contained elements required for phloem-specific expression while nucleotides −268 to −103 contained elements necessary for wound-specific expression. Interestingly, the main difference between the two CsSUS1p alleles was the presence of a 94-bp insertion in allele 2. Fusion of this indel to a minimal promoter and GUS reporter gene indicated that it contained stamen and carpel-specific enhancer elements. This finding of highly specific and separable regulatory units within the CsSUS1p suggests that this promoter may have a potential application in the generation of constructs for the use in the development of transgenic plants resistant to a wide variety of target pests.     

Tomato prosystemin promoter confers wound-inducible, vascular bundle-specific expression of the β-glucuronidase gene in transgenic tomato plants

Tomato (Lycopersicon esculentum Mill. cv. Better Boy) plants were transformed with a fused gene containing a 2.2-kb promoter fragment of the tomato prosystemin gene and the coding region of the β-glucuronidase (GUS) reporter gene. The transgenic plants exhibited a low constitutive level of prosystemin-β-glucuronidase gene expression, assayed by histochemical staining and GUS enzyme activity, that was associated in the vascular bundles of leaf main veins, petiolules, petioles and stems. The GUS activity in the vascular bundles in each tissue was increased by wounding and by treatment of the plants with methyl jasmonate, similar to the induction of prosystemin in wild-type plants. The increase in GUS activity in the vascular bundles of leaves in response to wounding correlated with the wound-inducible increase in prosystemin mRNA. Tissue printing, using rabbit anti-serum prepared against prosystemin, confirmed that inducible prosystemin protein was localized in vascular bundles of petiolules, petioles and stems of wild-type tomato plants. The evidence indicates that the 2.2-kb promoter region of the tomato prosystemin gene contains elements conferring its correct temporal and spatial expression in the vascular bundles of transgenic tomato plants. Received: 7 January 1997 / Accepted: 2 April 1997     

Tissue-specific expression of the β-glucuronidase reporter gene in transgenic strawberry (Fragaria × ananassa) plants

The strawberry ( Fragaria spp) is regarded as a false fruit because it originates from the receptacle, which is a non-ovarian tissue. For this reason, fruit-specific promoters isolated from plant species in which the fruit is derived from the ovary wall might not be suited to control gene expression in a fruit-specific way in strawberry. In order to achieve (false) fruit-specific expression in strawberry, we tested the petunia FBP7 (floral binding protein7) promoter, which proved to be active in the receptacles of petunia flowers, in transgenic strawberry fruits. In strawberry plants containing the FBP7 promoter fused to the ß-glucuronidase (GUS) reporter gene ( gus), GUS activity was found in floral and fruit tissues of all developmental stages tested but not in leaf, petiole and root tissue . Surprisingly, Northern blot analysis showed the presence of gus-derived mRNAs in root (strong) and petiole (weak) tissue of fbp7- gus plants in addition to the floral and fruit tissues. Therefore, it is concluded that the histological GUS phenotype does not necessarily correspond with expression at the mRNA level. mRNA quantification using the TaqMan polymerase chain reaction technology confirmed the Northern results and showed that in red strawberry tissue the cauliflower mosaic virus 35S promoter is at least sixfold stronger than the FBP7 promoter.     

The promoter of aBrassica napus polygalacturonase gene directs pollen expression ofβ-glucuronidase in transgenicBrassica plants

A 647-bp 5-flanking fragment obtained from genomic clone Sta 44G(2) belonging to a family of polygalacturonase genes expressed inBrassica napus pollen was fused to the-glucuronidase (GUS) marker gene. This fusion construct was introduced intoB. napus plants viaAgrobacterium tumefaciens transformation. Analysis of the transgenicB. napus plants revealed that this promoter fragment is sufficient to direct GUS expression specifically in the anther and that GUS activity increases in pollen during maturation.Abbreviation GUS -Glucuronidase     

Isolation of the promoter of a cotton β-galactosidase gene (GhGal1) and its expression in transgenic tobacco plants

β-galactosidases (GUS, EC 3.2.1.23) are character- ized by their ability to hydrolyze terminal, non-re- ducing β-D-galactosyl residues from β-D-galactosides and are widely distributed in microbes, plants and animals. To date, the primary structures of …     

Diversity and seasonal occurrence of mites (Acari) in a rubber tree crop (Hevea brasiliensis, Muell. Arg.) in northwestern São Paulo, Brazil

Brazilian southeastern region has soil and climate conditions suitable for the growing of rubber trees, and most part of national yield arises from S?o Paulo State. The aims proposed for this work were to determine the diversity, the richness and the seasonal occurrence of mites found in a rubber tree crop in a triennial survey with monthly samplings, as well as to estimate the populational density of the major phytophagous species. This study found 74,407 mites from 26 species belonging to 10 families. The phytophagous and predators represented 95.4% and 3.9% of the total abundance, respectively. Twelve species were rare, six accessories and eight constant. The families Phytoseiidae and Tydeidae had the greatest richness (five and four species, respectively). The most numerous species was Calacarus heveae Feres (50,573), with great abundance at the end of rainy season until the beginning of dry season. Among predators, the most abundant were Zetzellia quasagistemas Hernandes & Feres (1,345), Pronematus sp. (455), Zetzellia agistzellia Hernandes & Feres (409) and Euseius citrifolius Denmark & Muma (243). C. heveae had greatest densities on March and April 2003, and Lorryia formosa Cooreman and Tenuipalpus heveae Baker on March and May 2001, respectively. Many stigmaeids were observed in association with colonies of L. formosa preying their eggs and immatures.     

Mites (Acari) of rubber trees (Hevea brasiliensis Muell. Arg., Euphorbiaceae) in Piracicaba, state of São Paulo, Brazil

This study determined the main mite species on rubber trees (clone RRIM-600) in Piracicaba, southeast of S?o Paulo State, from June 2002 to May 2003 and evaluated the possible relation between them. It was conducted in a plantation of 5 ha on 11 year old trees, 15 m high, surrounded with crops as pearl millet, cotton, bean or corn. Samples were taken monthly and consisted of five leaflets, five petioles (only from October 2002 on) and five terminal sections of twigs (10 cm) from 15 rubber trees. All mites of one leaflet, one petiole and one twig section of each plant were mounted for identification to genera/species to estimate the proportional occurrence of each species. A total of 84,850 mites belonging to 38 species of 34 genera and 16 families were found. Tydeidae was the family with the highest number of species (11), followed by Phytoseiidae and Stigmaeidae (4 each). The most abundant families were Eriophyidae, Tenuipalpidae and Tydeidae (totals of 43,023, 26,390 and 13,644 individuals, respectively). The highest population levels of the pest mites Calacarus heveae Feres and Tenuipalpus heveae Baker occurred at the end of the rainy season. The most abundant predators were Metaseiulus camelliae (Chant & Yoshida-Shaul), Amblyseius compositus Denmark & Muma and Euseius citrifolius Denmark & Muma. The predators could not prevent the increase of C. heveae and T. heveae from March on. However, their presence might have prevented an earlier increase and even higher levels of those mites.     

Isolation of the β-carotene ketolase gene promoter from Haematococcus pluvialis and expression of ble in transgenic Chlamydomonas

The β-carotene ketolase gene (bkt1) is a key enzyme in the biosynthesis of astaxanthin in the green alga Haematococcus pluvialis. We constructed a genomic library of H. pluvialis from which the upstream sequence of bkt1 was cloned. It was just 27% identical to the β-C-4-oxygenase gene (crto1) promoter. A TATA-box and a number of CAAT-boxes were found in the bkt1 promoter region. Analysis of the sequence revealed the presence of cis-acting elements associated with light and stress-related responses. Seven novel GTAC core sequences involved in copper response were also detected. The bkt1 promoter was transferred into Chlamydomonas reinhardtii CC-849 to drive the expression of ble. The antibiotic resistance and expression of ble in Tran^BCO transgenic lines confirmed the promoter activity of the cloned bkt1 promoter sequence. The results of this study confirm that the bkt1 promoter owned cis-acting elements involved in light and environmental stresses and the genetic transformation system of C. reinhardtii can be used to study the functions of bkt1promoters from H. pluvialis.     

High-level expression of sucrose inducible sweet potato sporamin gene promoter: β-glucuronidase fusion gene in transgenic Nicotiana plumbaginifolia hairy roots

In Vitro Cellular & Developmental Biology - Plant - We developed transgenic Nicotiana plumbaginifolia hairy roots with sucrose-inducible minimal promoter (Spomin)-β-glucuronidase (GUS)...     

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.     

Mouse mammary tumor virus promoter directs high‐level expression of bovine αS1 casein in the milk of transgenic heterozygous and homozygous mice

Abstract

To test the mouse mammary tumor virus (MMTV) promoter as an inducible mammary‐specific promoter, we have produced 3 lines of transgenic mice carrying bovine αS1 casein cDNA under the control of MMTV promoter. The RNA analysis showed that in line 27, heterozygotes expressed 25%, and homozygotes 37% of the endogenous αSI casein mRNA of a mid‐lactation cow. Dexamethasone increased expression by about 3 fold in both heterozygotes and homozygotes. A similar increase in the level of mRNA was observed in both heterozygotes and homozygotes from line 42 with/without induction by dexamethasone. The transgenes were expressed predominantly in the mammary gland with low levels in the salivary gland, spleen, lung, and kidney. Their expression in mammary glands was lactation‐specific. The offspring from lines 27 and 42 expressed a high‐level bovine αS1 casein in their milk. Their expression in milk were 0.21 and 0.11 g/L for heterozygotes, 0.36 and 0.19 g/L for homozygotes, respectively. Dexamethasone further increased the levels of expression by approximately two fold for both heterozygotes and homozygotes.