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Characterization of a maize Wip1 promoter in transgenic plants.

ABSTRACT: The Maize Wip1 gene encodes a wound-induced Bowman-Birk inhibitor (BBI) protein which is a type of serine protease inhibitor, and its expression is induced by wounding or infection, conferring resistance against pathogens and pests. In this study, the maize Wip1 promoter was isolated and its function was analyzed. Different truncated Wip1 promoters were fused upstream of the GUS reporter gene and transformed into Arabidopsis, tobacco and rice plants. We found that (1) several truncated maize Wip1 promoters led to strong GUS activities in both transgenic Arabidopsis and tobacco leaves, whereas low GUS activity was detected in transgenic rice leaves; (2) the Wip1 promoter was not wound-induced in transgenic tobacco leaves, but was induced by wounding in transgenic rice leaves; (3) the truncated Wip1 promoter had different activity in different organs of transgenic tobacco plants; (4) the transgenic plant leaves containing different truncated Wip1 promoters had low GUS transcripts, even though high GUS protein level and GUS activities were observed; (5) there was one transcription start site of Wip1 gene in maize and two transcription start sites of GUS in Wip1::GUS transgenic lines; (6) the adjacent 35S promoter which is present in the transformation vectors enhanced the activity of the truncated Wip1 promoters in transgenic tobacco leaves, but did not influence the disability of truncated Wip1231 promoter to respond to wounding signals. We speculate that an ACAAAA hexamer, several CAA trimers and several elements similar to ACAATTAC octamer in the 5'-untranslated region might contribute to the strong GUS activity in Wip1231 transgenic lines, meanwhile, compared to the 5'-untranslated region from Wip1231 transgenic lines, the additional upstream open reading frames (uORFs) in the 5'-untranslated region from Wip1737 transgenic lines might contribute to the lower level of GUS transcript and GUS activity.

SUBMITTER: Zhang S

PROVIDER: S-EPMC3876083 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Characterization of a maize Wip1 promoter in transgenic plants.

Zhang Shengxue S Lian Yun Y Liu Yan Y Wang Xiaoqing X Liu Yunjun Y Wang Guoying G

International journal of molecular sciences 20131206 12

The Maize Wip1 gene encodes a wound-induced Bowman-Birk inhibitor (BBI) protein which is a type of serine protease inhibitor, and its expression is induced by wounding or infection, conferring resistance against pathogens and pests. In this study, the maize Wip1 promoter was isolated and its function was analyzed. Different truncated Wip1 promoters were fused upstream of the GUS reporter gene and transformed into Arabidopsis, tobacco and rice plants. We found that (1) several truncated maize Wip ...[more]

PMID: 24322445

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Project description:BackgroundGenetically modified plants are widely used in agriculture and increasingly in ecological research to enable the selective manipulation of plant traits in the field. Despite their broad usage, many aspects of unwanted transgene silencing throughout plant development are still poorly understood. A transgene can be epigenetically silenced by a process called RNA directed DNA methylation (RdDM), which can be seen as a heritable loss of gene expression. The spontaneous nature of transgene silencing has been widely reported, but patterns of acquirement remain still unclear.ResultsTransgenic wild tobacco plants (Nicotiana attenuata) expressing heterologous genes coding for antimicrobial peptides displayed an erratic and variable occurrence of transgene silencing. We focused on three independently transformed lines (PNA 1.2, PNA 10.1 and ICE 4.4) as they rapidly lost the expression of the resistance marker and down-regulated transgene expression by more than 200 fold after only one plant generation. Bisulfite sequencing indicated hypermethylation within the 35S and NOS promoters of these lines. To shed light on the progress of methylation establishment, we successively sampled leaf tissues from different stages during plant development and found a rapid increase in 35S promoter methylation during vegetative growth (up to 77% absolute increase within 45 days of growth). The levels of de novo methylation were inherited by the offspring without any visible discontinuation. A secondary callus regeneration step could interfere with the establishment of gene silencing and we found successfully restored transgene expression in the offspring of several regenerants.ConclusionsThe unpredictability of the gene silencing process requires a thorough selection and early detection of unstable plant lines. De novo methylation of the transgenes was acquired solely during vegetative development and did not require a generational change for its establishment or enhancement. A secondary callus regeneration step provides a convenient way to rescue transgene expression without causing undesirable morphological effects, which is essential for experiments that use transformed plants in the analysis of ecologically important traits.

Project description:Drought adversely affects crop growth and yields. The cloning and characterization of drought- or abscisic acid (ABA)-inducible promoters is of great significance for their utilization in the genetic improvement of crop resistance. Our previous studies have shown that maize sulfite oxidase (SO) has a sulfite-oxidizing function and is involved in the drought stress response. However, the promoter of the maize SO gene has not yet been characterized. In this study, the promoter (ZmSOPro, 1194 bp upstream region of the translation initiation site) was isolated from the maize genome. The in-silico analysis of the ZmSOPro promoter identified several cis-elements responsive to the phytohormone ABA and drought stress such as ABA-responsive element (ABRE) and MYB binding site (MBS), besides a number of core cis-acting elements, such as TATA-box and CAAT-box. A 5' RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmSO. The ZmSOPro activity was detected by β-glucuronidase (GUS) staining at nearly all developmental stages and in most plant organs, except for the roots in transgenic Arabidopsis. Moreover, its activity was significantly induced by ABA and drought stress. The 5'-deletion mutant analysis of the ZmSOPro in tobacco plants revealed that a 119-bp fragment in the ZmSOPro (upstream of the transcription start site) is a minimal region, which is required for its high-level expression. Moreover, the minimal ZmSOPro was significantly activated by ABA or drought stress in transgenic plants. Further mutant analysis indicated that the MBS element in the minimal ZmSOPro region (119 bp upstream of the transcription start site) is responsible for ABA and drought-stress induced expression. These results improve our understanding of the transcriptional regulation mechanism of the ZmSO gene, and the characterized 119-bp promoter fragment could be an ideal candidate for drought-tolerant gene engineering in both monocot and dicot crops.

Project description:BACKGROUND: Core promoters are cis-regulatory modules to which bind the basal transcriptional machinery and which participate in the regulation of transcription initiation. Although core promoters have not been extensively investigated through functional assays in a chromosomal context, the available data suggested that the response of a given core promoter might vary depending on the promoter context. Previous studies suggest that a (-57/+40) fragment constitutes the core promoter of the BhC4-1 gene which is located in DNA puff C4 of the sciarid fly Bradysia hygida. Here we tested this (-57/+40) fragment in distinct regulatory contexts in order to verify if promoter context affects its core promoter activity. RESULTS: Consistent with the activity of a core promoter, we showed that in the absence of upstream regulatory sequences the (-57/+40) fragment drives low levels of reporter gene mRNA expression throughout development in transgenic Drosophila. By assaying the (-57/+40) fragment in two distinct regulatory contexts, either downstream of the previously characterized Fbp1 enhancer or downstream of the UAS element, we showed that the BhC4-1 core promoter drives regulated transcription in both the germline and in various tissues throughout development. Furthermore, the use of the BhC4-1 core promoter in a UAS construct significantly reduced salivary gland ectopic expression in third instar larvae, which was previously described to occur in the context of the GAL4/UAS system. CONCLUSIONS: Our results from functional analysis in transgenic Drosophila show that the BhC4-1 core promoter drives gene expression regardless of the promoter context that was assayed. New insights into the functioning of the GAL4/UAS system in Drosophila were obtained, indicating that the presence of the SV40 sequence in the 3' UTR of a UAS construct does not preclude expression in the germline. Furthermore, our analysis indicated that ectopic salivary gland expression in the GAL4/UAS system does not depend only on sequences present in the GAL4 construct, but can also be affected by the core promoter sequences in the UAS construct. In this context, we propose that the sciarid BhC4-1 core promoter constitutes a valuable core promoter which can be employed in functional assays in insects.

Dataset Information

Characterization of a maize Wip1 promoter in transgenic plants.

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Characterization of a maize Wip1 promoter in transgenic plants.

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