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.     

Enhancing plant growth and biomass production by overexpression of GA20ox gene under control of a root preferential promoter

Overexpression of GA20 oxidase gene has been a recent trend for improving plant growth and biomass. Constitutive expression of GA20ox has successfully improved plant growth and biomass in several plant species. However, the constitutive expression of this gene causes side-effects, such as reduced leaf size and stem diameter, etc. To avoid these effects, we identified and employed different tissue-specific promoters for GA20ox overexpression. In this study, we examined the utility of At1g promoter to drive the expression of GUS (β-glucuronidase) reporter and AtGA20ox genes in tobacco and Melia azedarach. Histochemical GUS assays and quantitative real-time-PCR results in tobacco showed that At1g was a root-preferential promoter whose expression was particularly strong in root tips. The ectopic expression of AtGA20ox gene under the control of At1g promoter showed improved plant growth and biomass of both tobacco and M. azedarach transgenic plants. Stem length as well as stem and root fresh weight increased by up to 1.5–3 folds in transgenic tobacco and 2 folds in transgenic M. azedarach. Both tobacco and M. azedarach transgenic plants showed increases in root xylem width with xylem to phloem ratio over 150–200% as compared to WT plants. Importantly, no significant difference in leaf shape and size was observed between At1g::AtGA20ox transgenic and WT plants. These results demonstrate the great utility of At1g promoter, when driving AtGA20ox gene, for growth and biomass improvements in woody plants and potentially some other plant species.

     

Structural and functional analysis of new plant promoter pro-SmAMP1 from <Emphasis Type="Italic">Stellaria media</Emphasis>

The nucleotide sequence of a fragment of the promoter region of pro-SmAMP1 gene, having a length of 1257 bp and encoding antifungal peptides, was determined in chickweed (Stellaria media (L.) Vill.). Computer analysis of the nucleotide sequence revealed a number of cis-elements that are typical strong plant promoters. Five 5′-deletion variants were created taking into account the distribution of cis-elements:–1235,–771,–714,–603, and–481 bp of pro-SmAMP1 gene promoter, which were fused to the coding region of the uidA reporter gene in pCambia1381Z plant expression vector. The efficacy of pro-SmAMP1 promoter deletion variants was determined by transient expression in plants of Nicotiana benthamiana and using sequential generations of transgenic Nicotiana tabacum plants. It was found that the levels of GUS reporter protein activity in the extracts from transgenic and agroinfiltrated plants using all deletion variants of pro-SmAMP1 gene promoter were 3–5 times higher than those of 35S CaMV viral promoter. The highest activity of GUS protein was observed in the leaves of transgenic tobacco plants and closely correlated with the mRNA level of encoding gene. The levels of GUS activity did not differ significantly among 11 independent homozygous lines of T₂ generation of N. tabacum plants with different deletion variants of pro-SmAMP1 promoter. The results give reason to assume that all deletion variants of pro-SmAMP1 promoter provide stable and high level of expression of controlled genes. The shortest deletion variant–481 bp of pro-SmAMP1 promoter should be viewed as a potentially strong plant promoter for the genetic engineering of plants.     

Transgene expression in strawberries driven by a heterologous phloem-specific promoter

Strawberry is susceptible to diseases caused by phytoplasmas, mycoplasma-like prokaryotes restricted to sieve elements in the phloem tissue of infected plants. One strategy to improve strawberry resistance to phytoplasmas involves transgenic expression of anti-microbial peptide genes in phloem. For targeted phloem-specific expression, we constructed a binary vector with an expression cassette bearing the -glucuronidase (GUS) reporter gene (uidA) under control of the Arabidopsis sucrose-H⁺ symporter gene (AtSUC2) promoter. Transgenic strawberry lines were generated with high efficiencies by a modified transformation protocol, which combines the adoption of a 3-day pre-selection period following transformation, and the addition of 10-M thidiazuron to the regeneration medium. Histological GUS activity indicated that the reporter gene was expressed specifically in phloem of leaves, petioles, and roots of transgenic plants. The results suggest that the transformation protocol and the AtSUC2 promoter may be useful for engineering phytoplasma-resistant transgenic strawberries.     

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.     

Homologous recombination-mediated knock-in targeting of the MET1a gene for a maintenance DNA methyltransferase reproducibly reveals dosage-dependent spatiotemporal gene expression in rice

Although homologous recombination-promoted knock-in targeting to monitor the expression of a gene by fusing a reporter gene with its promoter is routine practice in mice, gene targeting to modify endogenous genes in flowering plants remains in its infancy. In the knock-in targeting, the junction sequence between a reporter gene and an endogenous target promoter can be designed properly, and transgenic plants carrying an identical and desired knock-in allele can be repeatedly obtained. By employing a reproducible gene-targeting procedure with positive–negative selection in rice, we were able to obtain fertile transgenic knock-in plants with the promoterless GUS reporter gene encoding β-glucuronidase fused with the endogenous promoter of MET1a , one of two rice MET1 genes encoding a maintenance DNA methyltransferase. All of the primary (T₀) transgenic knock-in plants obtained were found to carry only one copy of GUS , with the anticipated structure in the heterozygous condition, and no ectopic events associated with gene targeting could be detected. We showed the reproducible, dosage-dependent and spatiotemporal expression of GUS in the selfed progenies of independently isolated knock-in targeted plants. The results in knock-in targeted plants contrast sharply with the results in transgenic plants with the MET1a promoter -fused GUS reporter gene integrated randomly in the genome: clear interindividual variation of GUS expression was observed among independently obtained plants bearing the randomly integrated transgenes. As our homologous recombination-mediated gene-targeting strategy with positive–negative selection is, in principle, applicable to modify any endogenous gene, knock-in targeting would facilitate basic and applied plant research.     

A strong constitutive gene expression system derived from <Emphasis Type="Italic">ibAGP1</Emphasis> promoter and its transit peptide

To develop a strong constitutive gene expression system, the activities of ibAGP1 promoter and its transit peptide were investigated using transgenic Arabidopsis and a GUS reporter gene. The ibAGP1 promoter directed GUS expression in almost entire tissues including rosette leaf, inflorescence stem, inflorescence, cauline leaf and root, suggesting that the ibAGP1 promoter is a constitutive promoter. GUS expression mediated by ibAGP1 promoter was weaker than that by CaMV35S promoter in all tissue types, but when GUS protein was targeted to plastids with the aid of the ibAGP1 transit peptide, GUS levels increased to higher levels in lamina, petiole and cauline leaf compared to those produced by CaMV35S promoter. The enhancing effect of ibAGP1 transit peptide on the accumulation of foreign protein was tissue-specific; accumulation was high in lamina and inflorescence, but low in root and primary inflorescence stem. The transit peptide effect in the leaves was maintained highly regardless of developmental stages of plants. The ibAGP1 promoter and its transit peptide also directed strong GUS gene expression in transiently expressed tobacco leaves. These results suggest that the ibAGP1 promoter and its transit peptide are a strong constitutive foreign gene expression system for transgenesis of dicot plants.     

Expression of the peroxidase gene promoter (Shpx6b) from Stylosanthes humilis in transgenic plants during insect attack

Inducible promoters are important in regulating the expression of resistance genes when plants are attacked by insects or pathogens. Evaluation of the Shpx6b peroxidase promoter from the tropical forage legume Stylosanthes humilis[ Curtis MD, Rae AL, Rusu AG, Harrison SJ & Manners JM (1997) A peroxidase gene promoter induced by phytopathogens and methyl jasmonates in transgenic plants. Molecular Plant Microbial Interactions 10: 326–338] in transgenic tobacco plants Nicotiana tabacum L. (Solanaceae) demonstrated that this promoter could drive expression of both the β‐glucuronidase (GUS uidA gene of E. coli) and green fluorescent protein (GFP) reporter genes in leaf tissues during attack by chewing insects – larvae of potato tuber moth (PTM) Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae) and sucking insects – green peach aphids Myzus persicae Sulzer (Homoptera: Aphididae). Strong GUS expression was present in tissues next to cells damaged by PTM larvae 24 h after infestation. With aphid infestation, GUS expression was limited to sites of feeding, and was observed 48 h after infestation. The expression of GFP mirrored that of GUS expression for both treatments, but was normally detected 48 h after infestation. Similarly, the exogenous application of methyl jasmonate (MeJa) induced GUS uniformly across leaf tissue, and mechanical wounding activated GUS expression at wound sites, similar to PTM larvae. GFP expression was observed 48 h after treatment, and for mechanical wounding GFP was localised in a manner similar to PTM damage. For MeJa treatment, GFP expression was more pronounced in cells around the midrib, and it was not uniformly induced across the leaf tissue. GUS reporter gene levels were also assayed to quantify expression, and the results were consistent with the observed histological patterns of expression. The results presented here show that the Shpx6b promoter switches on the expression of linked genes after damage by insect herbivores, and could be useful in regulating the expression of heterologous genes for insect and/or pathogen resistance in transgenic plants.     

Visualization of somatic deletions mediated by R/<Emphasis Type="Italic">RS</Emphasis> site-specific recombination and induction of germinal deletions caused by callus differentiation and regeneration in rice

A transgenic rice plant expressing the recombinase of Zygosaccharomyces rouxii under the control of the CaMV 35S promoter was crossed with a transgenic plant carrying a cryptic (beta-glucuronidase) GUS reporter gene, which was activated by recombinase-mediated deletions between two specific recombination sites ( RSs). In F(1) plants, GUS activity was observed as blue spots and stripes in vascular bundles in several parts of the leaves. GUS expression was detected in all of the calli induced from F(1) seeds and throughout the regenerated plants. DNA analysis using the polymerase chain reaction and Southern blotting showed that R/ RS-mediated deletions occurred in all of the cells of the regenerated plants. Stable GUS expression was confirmed in the progeny resulting from self-pollination. Thus, the deletions obtained in the regenerated plants were genetically equivalent to the germinal deletions. These results indicate that the induction of callus differentiation and shoot regeneration is an effective manner to activate the R/ RS system and to produce plants with chromosomal deletions.     

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.     

Functional Characterisation of the Oil Palm Type 3 Metallothionein-like Gene (<Emphasis Type="Italic">MT3-B</Emphasis>) Promoter

In silico analysis showed that the differentially expressed type 3 oil palm metallothionein-like genes MT3-A and MT3-B share at least 11 common putative promoter regulatory elements. The identified motifs include W-boxes, TATCCA element, binding element for cytokinin response regulators and pollen-specific elements. A high degree of conservation was observed in their genomic organisation where the coding regions are divided at two identical positions in both genes by two AT-rich introns. Promoter activity of the MT3-B gene was analysed using a transient assay by bombarding oil palm tissue slices with a β-glucuronidase (GUS) gene construct and a stable reporter assay by analysing GUS expression in transformed Arabidopsis thaliana plants. Transient expression analysis revealed MT3-B promoter activity in oil palm root tissues but not in fruit mesocarp at 12 weeks after anthesis and spear leaves. The T3 homozygous transgenic Arabidopsis plants, harbouring the MT3-B promoter/GUS construct, showed reporter activity in cotyledons and mature leaves with lower expression levels in root tissues. The expression levels in the roots of the T3 homozygous transgenic plants increased five- and 2.5-folds when treated with 80 μM of Zn²⁺ and Fe²⁺, respectively. Altogether, these results indicate that the MT3-A and MT3-B promoter activities may be regulated by a variety of abiotic factors and MT3-B promoter may potentially be manipulated for use in plant genetic engineering for induced synthesis of gene product.     

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.     

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.