Efficiency of the tetracycline-dependent gene expression system: complete suppression and efficient induction of the rolB phenotype in transgenic plants

We have investigated the use of the tetracycline-dependent gene expression system to regenerate and propagate tobacco plants transformed with a gene whose product — when highly expressed — interferes with regeneration and/or further reproduction. Plants transformed with the Agrobacterium rhizogenes rolB gene under the control of the tetracycline-dependent expression system were phenotypically indistinguishable from wild type owing to efficient repression of the promoter. Induction of the rolB gene with tetracycline led to high-level expression of the rolB mRNA, which resulted in extremely stunted plants with necrotic and wrinkled leaves that did not develop a floral meristem. Upon cessation of tetracycline treatment healthy shoots developed even from severely affected meristems. Data on the dose response of the rolB phenotype as a function of tetracycline concentration demonstrate that the tetracycline-dependent gene expression system can be used to modulate the manifestation of a particular phenotype.     

Indole-3-acetic acid homeostasis in transgenic tobacco plants expressing the Agrobacterium rhizogenes rolB gene

The rolB gene of the plant pathogen Agrobacterium rhizogenes has an important role in the establishment of hairy root disease in infected plant tissues. When expressed as a single gene in transgenic plants the RolB protein gives rise to effects indicative of increased auxin activity. It has been reported that the RolB product is a β-glucosidase and proposed that the physiological and developmental alterations in transgenic plants expressing the rolB gene are the result of this enzyme hydrolysing bound auxins, in particular (indole-3-acetyl)-β-D-glucoside (IAGluc), and thereby bringing about an increase in the intracellular concentration of indole-3-acetic acid (IAA). Using tobacco plants as a test system, this proposal has been investigated in detail. Comparisons have been made between the RolB phenotype and that of IaaM/iaaH transformed plants overproducing IAA. In addition, the levels of IAA and IAA amide and IAA ester conjugates were determined in wild-type and transgenic 35S-rolB tobacco plants and metabolic studies were carried out with [¹³C₆]IAA [2′-¹⁴C]IAA, [¹⁴C]IAGluc, [5-³H]-2-o-(indole-3-acetyl)-myo-inositol and [¹⁴C]indole-3-acetylaspartic acid. The data obtained demonstrate that expression of the rolB encoded protein in transgenic tobacco does not produce a phenotype that resembles that of IAA over producing plants, does not alter the size of the free IAA pool, has no significant effect on the rate of IAA metabolism, and, by implication, appears not to influence the overall rate of IAA biosynthesis. Furthermore, the in vivo hydrolysis of IAGluc, and that of the other IAA conjugates that were tested, is not affected. On the basis of these findings, it is concluded that the RolB phenotype is not the consequence of an increase in the size of the free IAA pool mediated by an enhanced rate of hydrolysis of IAA conjugates.     

A recombinase-mediated transcriptional induction system in transgenic plants

We constructed and tested a Cre-loxP recombination-mediated vector system termed pCrox for use in transgenic plants. In this system, treatment of Arabidopsis under inducing conditions mediates an excision event that removes an intervening piece of DNA between a promoter and the gene to be expressed. The system developed here uses a heat-shock-inducible Cre to excise a DNA fragment flanked by lox sites, thereby generating a constitutive GUS reporter gene under control of the CaMV 35S promoter. Heat-shock-mediated excision of several, independent lines resulted in varying degrees of recombination-mediated GUS activation. Induction was shown to be possible at essentially any stage of plant growth. This single vector system circumvents the need for genetic crosses required by other, dual recombinase vector systems. The pCrox system may prove particularly useful in instances where transgene over-expression, or under-expression by antisense, would otherwise affect embryo, seed or seedling viability.     

New vector system for induction of gene expression in dicotyledonous plants

A system of two vectors, pEnLox and pCre, was developed. The pEnLox vector is used to induce insertional mutations, while pCre is used to obtain transgenic plants expressing the Cre recombinase gene. The T-DNA enhancer is flanked by the loxP sites in the pEnLox vector. As an Arabidopsis thaliana insertional mutant obtained by transformation with pEnLox is crossed with another transgenic line carrying the cre gene, the enhancer sequence is eliminated. The vector system makes it possible to induce insertional mutations and to determine whether the mutant phenotype is caused by the loss-of-function insertional mutation or by overespression of nearby genes in response to the enhancer contained in the insert.     

New vector system for induction of gene expression in dicotyledonous plants

A system of two vectors, pEnLox and pCre. was developed. The pEnLox vector is used to induce insertional mutations, while pCre is used to obtain transgenic plants expressing the Cre recombinase gene. The T-DNA enhancer is flanked by the loxP sites in the pEnLox vector. As an Arahidopsis thaliana insertional mutant obtained by transformation with pEnLox is crossed with another transgenic line carrying the cre gene. the enhancer sequence is eliminated. The vector system makes it possible to induce insertional mutations and to determine whether the mutant phenotype is caused by the loss-of-function insertional mutation or by overespression of nearby genes in response to the enhancer contained in the insert.     

Different promoter regions control level and tissue specificity of expression of Agrobacterium rhizogenes rolB gene in plants

Expression of the rolB gene of A. rhizogenes T-DNA triggers root differentiation in transformed plant cells. In order to study the regulation of this morphogenetic gene, the GUS reporter gene was placed under the control of several deleted fragments of the rolB 5 non-coding region: carrot disc transformations and the analysis of transgenic tobacco plants containing these constructions identified the presence of distinct regulatory domains in the rolB promoter. Two regions (located from positions –623 to –471 and from –471 to –341, from the translation start codon) control the level but not the tissue specificity of rolB expression: progressive deletions of the rolB promoter starting from position –1185 to –341, although at different levels, maintained the same pattern of GUS expression — maximal in root meristems and less pronounced in the vascular tissue of aerial organs. Further deletion of 35 bp, from –341 to –306, drastically affected tissue specificity: GUS activity was still clearly detectable in the vascular tissue of the aerial organs while expression in the root meristem was totally suppressed. Analysis of transgenic embryos and seedlings confirmed that distinct promoter domains are responsible for meristematic (root) and differentiated (vascular) expression of rolB. Finally, we present data concerning the effects of plant hormones on the expression of rolB-GUS constructions.     

转基因植物中报告基因gus的表达及其安全性评价

近十年来,植物转基因技术取得显著进展,许多转基因植物不断问世,其中报告基因ｇｕｓ在植物基因工程和遗传转化中发挥着重要的作用。本文就ｇｕｓ基因的来源、在转基因植物中的表达及其生态风险性与食品安全性作了简要综述。     

Variability of organ-specific gene expression in transgenic tobacco plants

Variability of expression of introduced marker genes was analysed in a large number of tobacco regenerants from anAgrobacterium-mediated transformation. In spite of standardization of sampling, considerable variation of GUS and NPTII expression was observed between individual transformants at different times of analysis and in different parts of the same plant. Organ-specificity of root versus leaf expression conferred by the par promoter from the haemoglobin gene ofParasponia andersonii in front of thegus gene showed a continuous spectrum. GUS expression in roots was found in 128 out of 140 plants; expression in leaves was found in 46 plants, and was always lower than in the corresponding roots. NPTII expression regulated by the nos promoter also showed a continuous spectrum. Expression levels were generally higher in roots than in leaves. Plants with high GUS expression in leaves showed high NPTII activity as well. A positive correlation between the level of NPTII expression and the numbers of integrated gene copies was noted. Chromosomal position effects and physiological determination are suggested as triggers for the variations. The transformed regenerated tobacco plants were largely comparable to clonal variants.     

Local and systemic induction of two defense-related subtilisin-like protease promoters in transgenic Arabidopsis plants. Luciferin induction of PR gene expression

Following a pathogenic attack, plants are able to mount a defense response with the coordinated activation of a battery of defense-related genes. In this study we have characterized the mode of expression of the P69B and P69C genes from tomato (Lycopersicon esculentum Mill.), which encodes two closely related subtilisin-like proteases associated with the defense response. We have compared the mode of gene regulation in heterologous transgenic Arabidopsis plants harboring promoter-beta-glucuronidase (GUS) and promoter-luciferase (LUC) gene fusions for these two genes. These studies revealed that the P69B and P69C promoters are induced by salicylic acid as well as during the course of both a compatible and an incompatible interaction with Pseudomonas syringae. Furthermore, P69B and P69C expression takes place in both the local and the distal (noninoculated) leaves upon inoculation with bacteria but following different and unique tissue-specific patterns of expression that are also different to that described for most other classical PR genes. Also, we report that luciferin, the substrate for the reporter luciferase (LUC) gene, is able to activate expression of PR genes, and this may pose a problem when using this gene reporter system in studies related to plant defense.     

Local induction of the alc gene switch in transgenic tobacco plants by acetaldehyde

The alc promoter system, derived from the filamentous fungi Aspergillus nidulans, allows chemically regulated gene expression in plants and thereby the study of gene function as well as metabolic and developmental processes. In addition to ethanol, this system can be activated by acetaldehyde, described as the physiological inducer in A. nidulans. Here, we show that in contrast to ethanol, acetaldehyde allows tissue-specific activation of the alc promoter in transgenic tobacco plants. Soil drenching with aqueous acetaldehyde solutions at a concentration of 0.05% (v/v) resulted in the rapid and temporary induction of the alc gene expression system exclusively in roots. In addition, the split root system allows activation to be restricted to the treated part of the root. The temporary activation of the alc system by soil drenching with acetaldehyde could be prolonged over several weeks by subsequent applications at intervals of 7 d. This effect was demonstrated for the root-specific induction of a yeast-derived apoplast-located invertase under the control of the alcohol-inducible promoter system. In leaves, which exhibit a lower responsiveness to acetaldehyde than roots, the alc system was induced in the directly treated tissue only. Thus, acetaldehyde can be used as a local inducer of the alc gene expression system in tobacco plants.     

Stable expression of promoterless bar gene in transgenic rape plants

Phosphinothricin resistant plants of two rapeseed (Brassica napus L. var. oleifera DC.) spring industrial cultivars were obtained by Agrobacterium tumefaciens leaf disk transformation. Vector constructions contained the promoterless coding sequence of phosphinothricin acetyltransferase (bar) gene located between two inverted lox-sites (elements of Cre/lox recombination system of P1 phage) and selective neomycinphosphotransferase II gene (nptII). Integration of the alien genes was confirmed by the PCR analyses. Stable and linked inheritance of foreign genes in T1 and T2 progeny was shown.     

Developmental regulation of expression of the malate synthase gene in transgenic plants

The cucumber malate synthase (MS) gene, including 1856 bp of 5 non-trnascribed sequence, has been transferred into Petunia (Mitchell) and Nicotiana plumbaginifolia plants using an Agrobacterium binary vector. The transferred gene is found in variable copy number in different transformants, and is stably transmitted in each case as a single Mendelian character. Transgene mRNA accumulates in the seedling during the first three days of germination, then declines in amount as the cotyledons emerge from the seed. The decline is more pronounced in light-grown seedlings than in dark-grown seedlings. Expression of the MS transgene is also detected at a low level in petals of transformed Petunia plants. In these respects the pattern of MS gene expression is similar in cucumber and in trnasformed plants, showing that the transferred DNA fragment contains a functional MS gene. A 1076 bp fragment of 5 sequence was linked to the -glucuronidase reporter gene and transferred into Nicotiana, where it was shown to direct temporal and spatial patterns of expression similar to that of the complete MS gene. However, histochemical localisation of -glucuronidase activity demonstrated that the chimaeric gene is expressed not only in cotyledons of transgenic plants, but also in endosperm and some hypocotyl cells during early germination. The relevance of these findings to the control of malate synthase gene expression is discussed.     

Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation

Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.