Functional characterization of a cotton late embryogenesis-abundant D113 gene promoter in transgenic tobacco

Previous studies have shown that mRNA and protein encoded by late embryogenesis-abundant (LEA) gene D113 from Gossypium hirsutum L. accumulate at high levels in mature seeds and also in response to abscisic acid (ABA) in young embryo. In this study, we studied the expression of four promoter 5′ deletion constructs (−1383, −974, −578 and −158) of the LEA D113 gene fused to beta-glucuronidase (GUS). GUS activity analysis revealed that the −578 promoter fragment was necessary to direct seed-specific GUS expression in transgenic tobacco plants (Nicotiana tabacum L.). To further investigate the expression pattern of LEA D113 promoter under environmental stresses, 2-week-old transgenic tobacco seedlings were exposed to ABA, dehydration, high salinity and cold treatments. GUS activity in the seedlings was quantified fluorimetrically, and expression was also observed by histochemical staining. An apparent increase in GUS activity was found in plants harboring constructs −1383, −974 and −578 after 24 h of ABA or high-salinity treatments, as well as after 10 days of dehydration. By contrast, only a slight increase was observed in all the three lines after cold treatment. Virtually no change in expression was found in construct −158 in response to dehydration, salinity and cold, but there was a moderate response to ABA, suggesting that the region between −574 and −158 was necessary for dehydration- and salinity-dependent expression, whereas ABA-responsive cis-acting elements might be located in the −158 region of the promoter.     

A novel wound-responsive cis-element, VWRE, of the vascular system-specific expression of a tobacco peroxidase gene, tpoxN1

The wound-induced expression of tpoxN1, encoding a tobacco peroxidase, is unique because of its vascular system-specific expression and insensitivity to known wound-signal compounds such as jasmonic acid, ethylene, and plant hormones [Sasaki et al. (2002) Plant Cell Physiol 43:108–117]. To study the mechanism of expression, the 2-kbp tpoxN1 promoter region and successive 5′-deletion of the promoter were introduced as GUS fusion genes into tobacco plants. Analysis of GUS activity in transgenic plants indicated that a vascular system-specific and wound-responsive cis-element (VWRE) is present at the −239/−200 region of the promoter. Gel mobility shift assays suggested that a nuclear factor(s) prepared from wounded tobacco stems binds a 14-bp sequence (−229/−215) in the −239/−200 region in a sequence-specific manner. A mutation in this 14-bp region of the −239 promoter fragment resulted in a considerable decrease in wound-responsive GUS activity in transgenic plants. An 11-bp sequence, which completely overlaps with the 14-bp sequence, was found in the 5′ distal region (−420/−410) and is thought to contribute to the wound-induced expression together with the 14-bp. The −114-bp core promoter of the tpoxN1 gene was indispensable for wound-induced expression, indicating that the 14-bp region is a novel wound-responsive cis-element VWRE, which may work cooperatively with other factors in the promoter.     

Development of a simple and efficient system for excising selectable markers in Arabidopsis using a minimal promoter::Cre fusion construct

The development of rapid and efficient strategies to generate selectable marker-free transgenic plants could help increase the consumer acceptance of genetically modified (GM) plants. To produce marker-free transgenic plants without conditional treatment or the genetic crossing of offspring, we have developed a rapid and convenient DNA excision method mediated by the Cre/loxP recombination system under the control of a −46 minimal CaMV 35S promoter. The results of a transient expression assay showed that −46 minimal promoter::Cre recombinase (−46::Cre) can cause the loxP-specific excision of a selectable marker, thereby connecting the 35S promoter and β-glucuronidase (GUS) reporter gene. Analysis of stable transgenic Arabidopsis plants indicated a positive correlation between loxP-specific DNA excision and GUS expression. PCR and DNA gel-blot analysis further revealed that nine of the 10 tested T₁ transgenic lines carried both excised and nonexcised constructs in their genomes. In the subsequent T₂ generation plants, over 30% of the individuals for each line were marker-free plants harboring the excised construct only. These results demonstrate that the −46::Cre fusion construct can be efficiently and easily utilized for producing marker-free transgenic plants.     

Functional characterization of the pollen-specific <Emphasis Type="Italic">SBgLR</Emphasis> promoter from potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

SBgLR (Solanum tuberosum genomic lysine-rich) gene was isolated from a potato genomic library using SB401 (S. berthaultii 401) cDNA as probe. RT-PCR analysis of SBgLR gene expression profile and microscopic analysis of green fluorescent protein (GFP) expression in tobacco plants transformed with SBgLR promoter-GFP reporters indicate that SBgLR is a pollen-specific gene. A series of 5′deletions of SBgLR promoter were fused to the β-glucuronidase (GUS) gene and stably introduced into tobacco plants. Histochemical and quantitative assays of GUS expression in transgenic plants allowed us to localize an enhancer of SBgLR promoter to the region −345 to −269 relative to the translation start site. This 76 bp (−345 to −269) fragment enhanced GUS expression in leaves, stems and roots when fused to −90/+6 CaMV 35S minimal promoter. Deletion analysis showed that a cis-element, which can repress gene expression in root hairs, was located in the region −345 to −311. Further study indicated that the −269 to −9 region was sufficient to confer pollen-specific expression of GFP when fused to CaMV 35S enhancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Authors Zhihong Lang and Peng Zhou contributed equally to this work.     

A 796?bp PsPR10 gene promoter fragment increased root-specific expression of the GUS reporter gene under the abiotic stresses and signal molecules in tobacco

A 1681 bp PsPR10 promoter was isolated from Pinus strobus and a series of 5′-deletions were fused to the β-glucuronidase (GUS) reporter gene and introduced into tobacco. GUS activity in P796 (−796 to +69) construct transgenic plant roots was similar with that of P1681 and higher than those of the P513 (−513 to +69) and P323 (−323 to +69) transgenic plants. Moreover, the abiotic stresses of NaCl, PEG 6000 and mannitol, and salicylic acid (SA), abscisic acid (ABA) and jasmonic acid (JA) induced higher GUS activity in the roots of P796 transgenic tobacco. This study provides a potential inducible root-specific promoter for transgenic plants.     

Pollen-Specific Expression of <Emphasis Type="Italic">Oryza sativa Indica</Emphasis> Pollen Allergen Gene (<Emphasis Type="Italic">OSIPA</Emphasis>) Promoter in Rice and <Emphasis Type="Italic">Arabidopsis</Emphasis> Transgenic Systems

Earlier, a pollen-specific Oryza sativa indica pollen allergen gene (OSIPA), coding for expansins/pollen allergens, was isolated from rice, and its promoter—upon expression in tobacco and Arabidopsis—was found active during the late stages of pollen development. In this investigation, to analyze the effects of different putative regulatory motifs of OSIPA promoter, a series of 5′ deletions were fused to β-glucuronidase gene (GUS) which were stably introduced into rice and Arabidopsis. Histochemical GUS analysis of the transgenic plants revealed that a 1631 bp promoter fragment mediates maximum GUS expression at different stages of anther/pollen development. Promoter deletions to −1272, −966, −617, and −199 bp did not change the expression profile of the pollen specificity. However, the activity of promoter was reduced as the length of promoter decreased. The region between −1567 and −199 bp was found adequate to confer pollen-specific expression in both rice and Arabidopsis systems. An approximate 4-fold increase in the GUS activity was observed in the pollen of rice when compared to that of Arabidopsis. As such, the OSIPA promoter seems promising for generation of stable male-sterile lines required for the production of hybrids in rice and other crop plants.     

Isolation of the maize <Emphasis Type="Italic">Zpu1</Emphasis> gene promoter and its functional analysis in transgenic tobacco plants

By screening a genomic library of maize, a 2.2 kb 5′ flanking fragment of Zpu1 gene, encoding the pullulanase-type starch debranching enzyme, was isolated. Promoter fragments of various lengths, including the full 5′ flanking sequence (−2267 to −1) (Z1), a 3′ deletion (−2267 to −513) (Z5) and three 5′ deletions extending to −1943 (Z2), −1143 (Z3) and −516 (Z4) upstream of the translational initiation codon (ATG), were fused to the GUS reporter gene and introduced into tobacco. When these constructs were tested in transgenic tobacco plants, seed-preferred GUS activity was observed in pZ1-transgenic lines. In pZ2-transgenic lines, the GUS activity was not only restricted to seeds, but was also detected in calyxes, petals, stamens and mature leaves. At the same time, negligible GUS activity was detected in roots, stems, young leaves, stigmas and ovaries from the transgenic tobacco plants, which had integrated the full isolated sequence of Zpu1 promoter or its deletions. Deletion analysis indicated that the promoter contained a putative positive cis-regulatory element and the proximal region (−516 to −1) was essential for directing the expression of gus reporter gene. Analysis of GUS activity during the fruit development and seed germination suggested that Zpu1 promoter is active both in starch anabolism and in starch catabolism, which is consistent with the function of the endogenous gene in maize. GUS activity in leaves under light and darkness confirmed that Zpu1 promoter functions in the starch degradation of photosynthetic tissues in the dark phase of the diurnal cycle.     

Authentic seed-specific activity of the <Emphasis Type="Italic">Perilla</Emphasis> oleosin 19 gene promoter in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The Perilla (Perilla frutescens L. cv. Okdong) oleosin gene, PfOle19, produces a 19-kDa protein that is highly expressed only in seeds. The activity of the −2,015 bp 5′-upstream promoter region of this gene was investigated in transgenic Arabidopsis plants using the fusion reporter constructs of enhanced green fluorescent protein (EGFP) and β-glucuronidase (GUS). The PfOle19 promoter directs Egfp expression in developing siliques, but not in leaves, stems or roots. In the transgenic Arabidopsis, EGFP fluorescence and histochemical GUS staining were restricted to early seedlings, indehiscent siliques and mature seeds. Progressive 5′-deletions up to the −963 bp position of the PfOle19 promoter increases the spatial control of the gene expression in seeds, but reduces its quantitative levels of expression. Moreover, the activity of the PfOle19 promoter in mature seeds is 4- and 5-fold greater than that of the cauliflower mosaic virus 35S promoter in terms of both EGFP intensity and fluorometric GUS activity, respectively.     

Effect of an enhanced CaMV 35S promoter and a fruit-specific promoter on uida gene expression in transgenic tomato plants

Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T₀ and T₁ plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T₁ plants segregated in a 3∶1 Mendelian ratio.     

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.     

Aryl hydrocarbon receptor (AhR)-mediated reporter gene expression systems in transgenic tobacco plants

In mammals, the aryl hydrocarbon receptor (AhR) mediates expression of certain genes, including CYP1A1, in response to exposure to dioxins and related compounds. We have constructed a mouse AhR-mediated gene expression systems for a β-glucuronidase (GUS) reporter gene consisting of an AhR, an AhR nuclear translocator (Arnt), and a xenobiotic response element (XRE)-driven promoter in transgenic tobacco plants. On treatment with the AhR ligands 3-methylcholanthrene (MC), β-naphthoflavone (βNF), and indigo, the transgenic tobacco plants exhibited enhanced GUS activity, presumably by inducible expression of the reporter gene. The recombinant AhR (AhRV), with the activation domain replaced by that of the Herpes simplex virus protein VP16, induced GUS activity much more than the wild-type AhR in the transgenic tobacco plants. Plants carrying AhRV expressed the GUS reporter gene in a dose- and time-dependent manner when treated with MC; GUS activity was detected at 5 nM MC on solid medium and at 12 h after soaking in 25 μM MC. Histochemical GUS staining showed that this system was active mainly in leaf and stem. These results suggest that the AhR-mediated reporter gene expression system has potential for the bioassay of dioxins in the environment and as a novel gene expression system in plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of a pollen-preferential gene, BAN102, from Chinese cabbage

We isolated and characterized a pollen-preferential gene, BAN102, from Chinese cabbage and analyzed the activity of its promoter. There were three or four copies of the BAN102 gene in the Chinese cabbage genome that specifically expressed in pollen and pollen tube. There were 2137 bp of BAN102 genomic clone comprising 186 bp of protein coding region, and 1178 bp of 5′ and 773 bp of 3′ non-coding regions. TATA box were located at 1071 nt of the promoter region while the polyadenylation signal and polyadenylation site were at 1470 and 1486 nt of the 3′ non-coding region. BLAST search of BAN102 sequence showed that coding region of BAN102 gene was the greatest percent similarity with arabinogalactan protein (AGP23) gene from Arabidopsis thaliana. Promoter analysis using GUS gene as a reporter showed that the pollen-specificity of BAN102 resided within the −112 to −44 bp of proximal promoter from the transient expression in tobacco and Chinese cabbage plants.     

Identification and preliminary analysis of a new PCP promoter from Brassica rapa ssp. chinensis

The promoter of Brassica campestris Male Fertile 5 (BcMF5), a pollen coat protein member, class A (PCP-A) gene family, was isolated from Brassica rapa L. ssp. chinensis Makino (Chinese cabbage-pak-choi) by Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR). Sequence analysis suggested that the 605-bp promoter of BcMF5 appears to be a pollen promoter. In an attempt to confirm the promoter activity of BcMF5 promoter, −609 to +3 bp and −377 to +3 bp fragments of the upstream sequence of BcMF5 were inserted at the site upstream of the coding region of the uidA gene in the sense orientation to construct two deletion expression vectors. Transient expression analysis in onion epidermal cells by particle bombardment showed that both −609 to +3 bp and −377 to +3 bp fragments of BcMF5 promoter were capable of driving β-glucuronidase gene expression. Furthermore, by Agrobacterium-mediated genetic transformation method, Arabidopsis transgenic Kan^R plants were obtained. GUS assay analysis revealed that the promoter of BcMF5 induced gene expression at the early stage of anther development and drove high levels of GUS expression in anther walls, upper regions of petals, pollen, and pollen tubes in the middle and late stage of anther development, but did not drive any expression in sepals and pistils.     

Multiple elements of the S 2 ?-RNase promoter from potato ( Solanum tuberosum L.) are required for cell type-specific expression in transgenic potato and tobacco

A functional analysis of the promoter of the S ₂ -RNase gene from potato was performed in transgenic potato and tobacco plants, using a deletion series of S ₂ -RNase promoter GUS fusions. A detailed histochemical and quantitative analysis of the transgenic tobacco plants revealed that S ₂ promoter fragments ranging in size from 5.6 kb in length down to 0.2 kb mediate a weak developmentally regulated expression in the pistil, and strong ectopic expression in pollen. In the pistil, different expression patterns were seen depending on the transformant, the predominant one being characterised by expression in the stigma and the transmitting tract of the style, whereas a few plants showed expression exclusively either in the stigma or in the stylar transmitting tissue. All transformants also showed GUS expression in the placental epidermis of the ovary. Two sequences that are conserved between the potato S ₁ -RNase and S ₂ -RNase promoters, termed motif I and motif III, are located in a fragment of the S ₂ promoter extending from position −200 to bp −100, and motif II, located between bp −498 and −480, was identified on the basis of sequence comparisons between pistil-specific promoters. Motif II was found to be dispensible for pistil-specific and for pollen-specific expression. Two submotifs, A and B, were identified within motif I. Both were essential for expression in the pistil but only B was necessary for expression in pollen. Although motif III has a similar bipartite structure and sequence to motif I, it was not sufficient to confer either pollen- or pistil-specific expression. However, deletion of motif III abolished pollen-specific expression in transient expression experiments, suggesting that an interaction between the two sequence motifs may be needed to specify cell type-specific expression. In transgenic potato the S ₂ -RNase promoter also mediates expression in pollen and in the pistil; however, significantly fewer plants showed expression than in tobacco, with most plants also exhibiting GUS expression in other tissues. Received: 7 August 1997 / Accepted: 8 September 1997