Activate Reporter Gene Expression Research Articles

IE1 of Autographa californica Multiple Nucleopolyhedrovirus Activates Low Levels of Late Gene Expression in the Absence of Virus RNA Polymerase.

Early and late gene expressions of baculoviruses have been known to rely on host RNA polymerase II and a virus-encoded RNA polymerase, separately. In this study, we found that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) recombinant bacmids with the individual RNA polymerase subunit genes deleted could support low levels of expression of a reporter gene under the control of the promoter of a typical late gene, vp39, in transfected Sf9 cells. Through multistep subcloning of a genomic library of the virus and transient expression assay analysis, ie1 was identified to be the only viral gene that was responsible for activation of late gene expression in the absence of the viral RNA polymerase. Furthermore, IE1 was found to be capable of activating reporter gene expression from the promoters of additional late genes polh, p6.9, odv-e18, odv-e25, and gp41, independent of any additional viral factors. Deletion of ie1 from the virus genome eliminated late gene expression. The IE1-activated late gene expression was enhanced by the viral hr4b. It was shown to be insensitive to inhibition of α-amanitin and did not appear to have stable transcription start sites. It is proposed that IE1 may serve to recruit newly synthesized viral RNA polymerase to viral DNA by activating low levels of pretranscription of the late genes to create an appropriate DNA conformation. IMPORTANCE The late gene expression of baculovirus has been known to depend on the virus-encoded RNA polymerase, which consists of four subunits. The immediate-early gene ie1 was found to be required for viral early gene expression, late gene expression, and DNA replication. How it functions in late gene expression remains unclear. In this study, we found that AcMNPV IE1 could activate low levels of gene expression from late gene promoters independently of any additional viral factors, with nonspecific transcription start sites. This new finding will shed light on the role of IE1 in the regulation of late gene expression and the understanding of the mechanism of late gene transcription initiation.

Membrane and Nuclear Yeast Two-Hybrid Systems.

The yeast two-hybrid (Y2H) system is an effective means of detecting protein-protein interactions through the activation of reporter gene expression. This system has also displayed several useful applications, including rapidly revealing unexpected interactors of known proteins, detecting regions that take part in protein-protein interactions, and characterizing the orchestration of protein interactions in metabolic pathways. The Y2H system has been widely utilized in the study of plant virology to investigate interactions between viral proteins and host, vector, or viral proteins. This technology has been successful in enhancing the understanding of viral structure and assembly, replication, viral gene expression and regulation, viral movement, pathogenicity, and the network of viral proteins. Here, we provide the methods to uncover novel interactors of viral proteins via screening cDNA libraries of plant host and insect vectors, using nuclear and membrane Y2H systems, respectively.

Unusual DNA-binding properties of the Arabidopsis thaliana WRKY50 transcription factor at target gene promoters

Key messageWRKY50 from A. thaliana requires WT-boxes at target gene promoters for activation and binding.Based on the genome-wide prediction of WRKY50 target genes and the similarity of a WRKY50 binding site to WT-boxes in microbe-associated molecular pattern (MAMP)-responsive cis-regulatory modules (CRM), four WT-box containing CRMs from the promoter region of three WRKY50 target genes were investigated for their interaction with WRKY50. These target genes are DJ1E, WRKY30 and ATBBE4. Two of the four CRMs, one from DJ1E and one from WRKY30, were able to activate reporter gene expression in the presence of WRKY50. Activation requires the WT-boxes GGACTTTT, GGACTTTG from DJ1E and GGACTTTC from WRKY30. WRKY50 does not activate a second CRM from WRKY30 and the CRM from ATBBE4, both containing the WT-box TGACTTTT. In vitro gel-shift assays demonstrate WT-box-specific binding of the WRKY50 DNA-binding domain to all four CRMs. This work shows a high flexibility of WRKY50 binding site recognition beyond the classic W-box TTGACC/T.

Optimization of a bacterial‐three hybrid assay for discovery and characterization of sRNA‐binding proteins

Non‐coding RNAs regulate gene expression in every domain of life. In bacteria, small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by protein chaperones. Hfq is an RNA chaperone protein that, through RNA interactions on distinct surfaces of its hexameric structure, supports many sRNAs to regulate translation and stability of their mRNA targets. Though Hfq is well characterized in E. coli and some additional model bacteria, Hfq homologs have only been identified in ~50% of sequenced bacteria, leading to speculation that additional RNA chaperone proteins may play important roles in sRNA function in other bacteria. We have recently developed a bacterial three‐hybrid (B3H) assay that detects the binding of E. coli sRNAs with multiple RNA chaperone proteins. This assay couples the transcription of a genetic reporter to the interaction of a DNA bound “bait” RNA (e.g. an sRNA) and an RNAP‐fused “prey” protein (e.g. Hfq). Successful interaction between RNA and protein stabilizes RNA polymerase at a test promoter and activates reporter gene expression. While this assay current detects numerous high‐affinity RNA‐protein interactions, the signal‐to‐noise makes it challenging to detect weaker interactions. In order to lay the foundation to search for new bacterial RNA‐binding proteins, we are using Hfq‐sRNA interactions as a model system to optimize the system, so that weaker RNA‐protein interactions can be reliably detected. Here we will present our efforts to improve the assay’s signal‐to‐noise ratio by adjusting the sequence, concentration, and spacing of different components within the B3H assay. With these improvements, we will work to identify novel protein‐RNA interactions, as well as dissect the binding mechanisms of these interactions.Support or Funding InformationThis work was supported by National Institutes of Health [R15GM135878], Mount Holyoke College and the Henry Luce foundation.

A WRKY transcription factor, FtWRKY46, from Tartary buckwheat improves salt tolerance in transgenic Arabidopsis thaliana

The WRKY transcription factor family includes plant-specific transcription factors that are widely involved in plant biotic and abiotic stress responses, growth and development. Tartary buckwheat is a type of small grain with strong resistance to adverse growing conditions. No systematic exploration of the WRKY family in Tartary buckwheat has yet been reported. In this paper, we report the FtWRKY46 gene from Tartary buckwheat and study its role in salt tolerance. FtWRKY46 has transcriptional activation activity in yeast, and FtWRKY46 fused to yellow fluorescent protein localizes to the nucleus. Further studies have found that its transcriptional activation region is located at the N-terminus. A yeast one-hybrid assay indicated that FtWRKY46 could bind to a W-box and activate reporter gene expression. Similarly, transient cotransfection showed that FtWRKY46 could specifically bind to W-box regions and activate reporter gene expression in plants. Furthermore, ectopic expression of FtWRKY46 could enhance Arabidopsis tolerance to salt stress. More specifically, the seed germination rate, root length, chlorophyll content and proline content were significantly higher in transgenic plants ectopically expressing FtWRKY46 than in WT plants after salt stress (P < 0.05), while MDA levels were significantly lower than in WT plants (P < 0.05). Additionally, salt treatment increased the expression of stress-related genes. To summarize, our results suggest that ectopic expression of FtWRKY46 enhance the stress tolerance of transgenic plants by modulating ROS clearance and stress-related gene expression.

The functional identification of glycine-rich TtASR from Tetragonia tetragonoides (Pall.) Kuntze involving in plant abiotic stress tolerance

In this study, we reported on an ASR gene (TtASR) related to salt/drought tolerance from the edible halophyte Tetragonia tetragonoides (Pall.) Kuntze (Aizoaceae). A phylogenetic analysis revealed that TtASR was evolutionarily close to other two halophytic glycine-rich ASR members, SbASR-1 (from Salicornia brachiate) and SlASR (from Suaeda liaotungensis), with a typical abscisic acid (ABA)/water-deficit stress (WDS) domain at C-terminal. Quantitative RT-PCR analyses showed that TtASR was expressed in all tested different organs of the T. tetragonoides plant and that expression levels were apparently induced after salt, osmotic stress, and ABA treatments in T. tetragonoides seedlings. An induction of TtASR improved the growth performance of yeast and bacteria more than the control under high salinity, osmotic stress, and oxidative stress. TtASR was not a nuclear-specific protein in plant, and the transcriptional activation assay also demonstrated that TtASR could not activate reporter gene's expression in yeast. TtASR overexpressed Arabidopsis plants exhibited higher tolerance for salt/drought and oxidative stresses and lower ROS accumulation than wild type (WT) plants, accompanied by increased CAT, SOD activities, higher proline content, and lower MDA content in vivo. The results indicated that the TtASR was involved in plant responses to salt and drought, probably by mediating water homeostasis or by acting as ROS scavengers, and that it decreased the membrane damage and improved cellular osmotic adjustment that respond to abiotic stresses in microorganisms and plants.

Development of a Gateway-compatible two-component expression vector system for plants.

Genetic transformation of plants offers the possibility of functional characterization of individual genes and the improvement of plant traits. Development of novel transformation vectors is essential to improve plant genetic transformation technologies for various applications. Here, we present the development of a Gateway-compatible two-component expression vector system for Agrobacterium-mediated plant transformation. The expression system contains two independent plasmid vector sets, the activator vector and the reporter vector, based on the concept of the GAL4/UAS trans-activation system. The activator vector expresses a modified GAL4 protein (GAL4-VP16) under the control of specific promoter. The GAL4-VP16 protein targets the UAS in the reporter vector and subsequently activates reporter gene expression. Both the activator and reporter vectors contain the Gateway recombination cassette, which can be rapidly and efficiently replaced by any specific promoter and reporter gene of interest, to facilitate gene cloning procedures. The efficiency of the activator-reporter expression system has been assessed using agroinfiltration mediated transient expression assay in Nicotiana benthamiana and stable transgenic expression in Arabidopsis thaliana. The reporter genes were highly expressed with precise tissue-specific and subcellular localization. This Gateway-compatible two-component expression vector system will be a useful tool for advancing plant gene engineering.

Optimization of a Bacterial Three‐Hybrid Assay for RNA‐Protein Interactions

Non‐coding RNAs regulate gene expression in every domain of life. In bacteria, non‐coding small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by protein chaperones. While much is known about the mechanism of a few model RNA chaperone proteins, significant gaps remain in our understanding of how other bacterial RNA‐binding proteins interact with RNA to regulate gene expression. While there are numerous biochemical methods to study bacterial RNA‐protein interactions, new genetic methodologies would be a powerful complement to current tools. We have recently developed a bacterial three‐hybrid (B3H) assay that detects the binding of E. coli sRNAs with multiple RNA chaperone proteins. This assay couples the transcription of a genetic reporter to the interaction of a DNA‐bound “bait” RNA (e.g. an sRNA) and an RNAP‐fused “prey” protein (e.g. Hfq). Successful interaction between RNA bait and protein prey stabilizes RNA polymerase (RNAP) at a test promoter and activates reporter gene expression. This B3H assay offers a straightforward path to identify and interrogate mutations in these RNA‐binding proteins with molecular phenotypes of interest. Currently, only ~40% of known sRNA‐chaperone interactions are detectable in the system. In order to maximize the utility of our bacterial three‐hybrid assay, a larger proportion of known RNA‐protein interactions must be successfully detectable. Current efforts are focused on optimizing the signal of the B3H assay so that weaker RNA‐protein interactions can be reliably detected. Here, we show results from our efforts to improve the assay's signal‐to‐noise ratio by tuning the sequences, spacing, and the concentrations of the B3H assay components. We have observed an improvement in the assay's signal in response to systematic alterations of the assay components. With an optimized B3H assay, we aim to discover new sRNA‐binding proteins and uncover the regulatory roles sRNAs and these proteins play in biology.Support or Funding InformationMount Holyoke College and the Luce FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transcription factors involved in basal immunity in mammals and plants interact with the same MAMP-responsive cis-sequence from Arabidopsis thaliana.

WRKY and NF-κB transcription factors, involved in innate immunity in plants and mammals, interact with the same cis-sequence. Novel microbe-associated molecular pattern (MAMP)-responsive cis-sequences, designated type II WT-boxes, are required for flg22-responsive gene expression in Arabidopsis thaliana protoplasts. While type I WT-boxes like TGACTTTT and CGACTTTT interact with WRKY transcription factors (TFs), the question remained which TFs bind to the type II WT-boxes GGACTTTC, GGACTTTT, and GGACTTTG. Surprisingly, a bioinformatic analysis predicts mouse (Mus musculus) NF-κB p65 as a TF interacting with type II WT-boxes. NF-κB p65, like WRKY factors in plants, plays a role in innate immunity in mammals. Therefore, the interaction of NF-κB p65 with type II WT-boxes was tested experimentally. NF-κB p65 requires the WT-boxes GGACTTTC, GGACTTTT, and GGACTTTG for activating reporter gene expression in plant cells. NF-κB p65 directly binds to WT-box containing synthetic promoters in vitro and requires the WT-box for binding. Earlier studies indicate that the sequence GGACTTTC is also required for WRKY26 mediated reporter gene activation. Here it is shown that WRKY26, like NF-κB p65, binds to the sequence GGACTTTC. Consistent with other recent studies, type II WT boxes are WRKY binding sites and the distinction between type I and type II no longer applies.

Targeted deletion of an NRL- and CRX-regulated alternative promoter specifically silences FERM and PDZ domain containing 1 (Frmpd1) in rod photoreceptors.

Regulation of cell type-specific gene expression is critical for generating neuronal diversity. Transcriptome analyses have unraveled extensive heterogeneity of transcribed sequences in retinal photoreceptors because of alternate splicing and/or promoter usage. Here we show that Frmpd1 (FERM and PDZ domain containing 1) is transcribed from an alternative promoter specifically in the retina. Electroporation of Frmpd1 promoter region, -505 to +382bp, activated reporter gene expression in mouse retina in vivo. A proximal promoter sequence (-8 to +33bp) of Frmpd1 binds to neural retina leucine zipper (NRL) and cone-rod homeobox protein (CRX), two rod-specific differentiation factors, and is necessary for activating reporter gene expression in vitro and in vivo. Clustered regularly interspaced short palindromic repeats/Cas9-mediated deletion of the genomic region, including NRL and CRX binding sites, in vivo completely eliminated Frmpd1 expression in rods and dramatically reduced expression in rod bipolar cells, thereby overcoming embryonic lethality caused by germline Frmpd1 deletion. Our studies demonstrate that a cell type-specific regulatory control region is a credible target for creating loss-of-function alleles of widely expressed genes.

An NAC domain transcription factor ATAF2 acts as transcriptional activator or repressor dependent on promoter context.

The ARABIDOPSIS THALIANA ACTIVATION FACTOR 2 (ATAF2) protein has been demonstrated to be involved in various biological processes including biotic stress responses, photo morphogenesis, and auxin catabolism. However, the transcriptional function of ATAF2 currently remains elusive. Therefore, to further understand the molecular function of ATAF2, we evaluated the transcriptional activities of ATAF2 using a transient assay system in this study. We used an effector consisting of a GAL4-DNA binding domain (GAL4-BD) fused to ATAF2, and observed upregulated reporter gene expression, suggesting that ATAF2 potentially has transcriptional activation activity. ATAF2 has been shown to activate reporter gene expression under the control of the ORE1 promoter. By contrast, ATAF2 significantly repressed reporter gene expression driven by the NIT2 promoter. These data suggest that ATAF2 is a bifunctional transcription factor that can alter target gene expression depending on the promoter sequences.

Engineered Biosensors from Dimeric Ligand-Binding Domains.

Biosensors are important components of many synthetic biology and metabolic engineering applications. Here, we report a second generation of Saccharomyces cerevisiae digoxigenin and progesterone biosensors based on destabilized dimeric ligand-binding domains that undergo ligand-induced stabilization. The biosensors, comprising one ligand-binding domain monomer fused to a DNA-binding domain and another fused to a transcriptional activation domain, activate reporter gene expression in response to steroid binding and receptor dimerization. The introduction of a destabilizing mutation to the dimer interface increased biosensor dynamic range by an order of magnitude. Computational redesign of the dimer interface and functional selections were used to create heterodimeric pairs with further improved dynamic range. A heterodimeric biosensor built from the digoxigenin and progesterone ligand-binding domains functioned as a synthetic "AND"-gate, with 20-fold stronger response to the two ligands in combination than to either one alone. We also identified mutations that increase the sensitivity or selectivity of the biosensors to chemically similar ligands. These dimerizing biosensors provide additional flexibility for the construction of logic gates and other applications.

Functional analysis of the HD-Zip transcription factor genes Oshox12 and Oshox14 in rice.

The homeodomain-leucine zipper (HD-Zip) transcription factor family plays vital roles in plant development and morphogenesis as well as responses to biotic and abiotic stresses. In barley, a recessive mutation in Vrs1 (HvHox1) changes two-rowed barley to six-rowed barley, which improves yield considerably. The Vrs1 gene encodes an HD-Zip subfamily I transcription factor. Phylogenetic analysis has shown that the rice HD-Zip I genes Oshox12 and Oshox14 are the closest homologues of Vrs1. Here, we show that Oshox12 and Oshox14 are ubiquitously expressed with higher levels in developing panicles. Trans-activation assays in yeast and rice protoplasts demonstrated that Oshox12 and Oshox14 can bind to a specific DNA sequence, AH1 (CAAT(A/T)ATTG), and activate reporter gene expression. Overexpression of Oshox12 and Oshox14 in rice resulted in reduced panicle length and a dwarf phenotype. In addition, Oshox14 overexpression lines showed a deficiency in panicle exsertion. Our findings suggest that Oshox12 and Oshox14 may be involved in the regulation of panicle development. This study provides a significant advancement in understanding the functions of HD-Zip transcription factors in rice.

Detecting Interactions of Membrane Proteins: The Split-Ubiquitin System.

The in vivo analysis of protein-protein interactions (PPIs) is a critical factor for gaining insights into cellular mechanisms and their biological functions. To that end, a constantly growing number of genetic tools has been established, some of which are using baker's yeast (Saccharomyces cerevisiae) as a model organism. Here, we provide a detailed protocol for the yeast mating-based split-ubiquitin system (mbSUS) to study binary interactions among or with full-length membrane proteins in their native subcellular environment. The system is based on the reassembly of two autonomously non-functional ubiquitin moieties attached to proteins of interest (POIs) into a native-like molecule followed by the release of a transcription factor. Upon its nuclear import, the activation of reporter gene expression gives a visual output via growth on interaction-selective media. Additionally, we apply a modification of the classical split-ubiquitin technique called CytoSUS that detects interactions of non-membrane/soluble proteins in their full-length form via translational fusion of an ER membrane anchor.

Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex.

Med6 protein (Med6p) is a hallmark component of evolutionarily conserved Mediator complexes, and the genuine role of Med6p in Mediator functions remains elusive. For the functional analysis of Saccharomyces cerevisiae Med6p (scMed6p), we generated a series of scMed6p mutants harboring a small internal deletion. Genetic analysis of these mutants revealed that three regions (amino acids 33–42 (Δ2), 125–134 (Δ5), and 157–166 (Δ6)) of scMed6p are required for cell viability and are located at highly conserved regions of Med6 homologs. Notably, the Med6p-Δ2 mutant was barely detectable in whole-cell extracts and purified Mediator, suggesting a loss of Mediator association and concurrent rapid degradation. Consistent with this, the recombinant forms of Med6p having these mutations partially (Δ2) restore or fail (Δ5 and Δ6) to restore in vitro transcriptional defects caused by temperature-sensitive med6 mutation. In an artificial recruitment assay, Mediator containing a LexA-fused wild-type Med6p or Med6p-Δ5 was recruited to the lexA operator region with TBP and activated reporter gene expression. However, the recruitment of Mediator containing LexA-Med6p-Δ6 to lexA operator region resulted in neither TBP recruitment nor reporter gene expression. This result demonstrates a pivotal role of Med6p in the postrecruitment function of Mediator, which is essential for transcriptional activation by Mediator.

Reporter Analyses Reveal Redundant Enhancers that Confer Robustness on Cis-Regulatory Mechanisms.

Reporter analyses of Hox1 and Brachyury (Bra) genes have revealed examples of redundant enhancers that provide regulatory robustness. Retinoic acid (RA) activates through an RA-response element the transcription of Hox1 in the nerve cord of the ascidian Ciona intestinalis. We also found a weak RA-independent neural enhancer within the second intron of Hox1. The Hox1 gene in the larvacean Oikopleura dioica is also expressed in the nerve cord. The O. dioica genome, however, does not contain the RA receptor-encoding gene, and the expression of Hox1 has become independent of RA. We have found that the upstream sequence of the O. dioica Hox1 was able to activate reporter gene expression in the nerve cord of the C. intestinalis embryo, suggesting that an RA-independent regulatory system in the nerve cord might be common in larvaceans and ascidians. This RA-independent redundant regulatory system may have facilitated the Oikopleura ancestor losing RA signaling without an apparent impact on Hox1 expression domains. On the other hand, vertebrate Bra is expressed in the ventral mesoderm and notochord, whereas its ascidian ortholog is exclusively expressed in the notochord. Fibroblast growth factor (FGF) induces Bra in the ventral mesoderm in vertebrates, whereas it induces Bra in the notochord in ascidians. Disruption of the FGF signal does not completely silence Bra expression in ascidians, suggesting that FGF-dependent and independent enhancers might comprise a redundant regulatory system in ascidians. The existence of redundant enhancers, therefore, provides regulatory robustness that may facilitate the acquisition of new expression domains.

Sequence and functional characterization of hypoxia-inducible factors, HIF1α, HIF2αa, and HIF3α, from the estuarine fish, Fundulus heteroclitus.

The hypoxia-inducible factor (HIF) family of transcription factors plays central roles in the development, physiology, pathology, and environmental adaptation of animals. Because many aquatic habitats are characterized by episodes of low dissolved oxygen, fish represent ideal models to study the roles of HIF in the response to aquatic hypoxia. The estuarine fish Fundulus heteroclitus is found in habitats prone to hypoxia. It responds to low oxygen via behavioral, physiological, and molecular changes, and one member of the HIF family, HIF2α, has been previously described. Herein, cDNA sequencing, phylogenetic analyses, and genomic approaches were used to determine other members of the HIFα family from F. heteroclitus and their relationships to HIFα subunits from other vertebrates. In vitro and cellular approaches demonstrated that full-length forms of HIF1α, HIF2α, and HIF3α independently formed complexes with the β-subunit, aryl hydrocarbon receptor nuclear translocator, to bind to hypoxia response elements and activate reporter gene expression. Quantitative PCR showed that HIFα mRNA abundance varied among organs of normoxic fish in an isoform-specific fashion. Analysis of the F. heteroclitus genome revealed a locus encoding a second HIF2α-HIF2αb-a predicted protein lacking oxygen sensing and transactivation domains. Finally, sequence analyses demonstrated polymorphism in the coding sequence of each F. heteroclitus HIFα subunit, suggesting that genetic variation in these transcription factors may play a role in the variation in hypoxia responses among individuals or populations.

Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast.

Whole-cell biocatalysts have proven a tractable path toward sustainable production of bulk and fine chemicals. Yet the screening of libraries of cellular designs to identify best-performing biocatalysts is most often a low-throughput endeavor. For this reason, the development of biosensors enabling real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily of LysR-type transcriptional regulators (LTTRs). We identified a design supporting LTTR-dependent activation of reporter gene expression in the presence of cognate small-molecule inducers. As proof of principle, we applied the biosensors for in vivo screening of cells producing naringenin or cis,cis-muconic acid at different levels, and found that reporter gene output correlated with production. The transplantation of prokaryotic transcriptional activators into the eukaryotic chassis illustrates the potential of a hitherto untapped biosensor resource useful for biotechnological applications.

Identification and functional characterisation of a novel anther-specific LTP promoter from Brassica campestris ssp. chinensis

ABSTRACTThe pollen development-related gene msLTP from Brassica campestris ssp. chinensis belongs to the lipid transfer protein (LTP) gene family. The promoter of the msLTP gene was isolated by thermal asymmetric interlaced PCR (TAIL-PCR). The functional elements of the promoter (named Bc15) were analysed. Some anther/pollen-specific elements, such as the GGTT box and the GTGA box, were identified in the sequence. In an attempt to confirm the promoter activity, a series of 5′-truncated constructs was fused with the GUS reporter gene to functionally analyse the key regulatory regions of the promoter. Transient expression analysis showed high GUS activity in onion epidermal cells containing the region from −731 to −1 bp. The truncated construct bearing the −731 to −1 bp fragment was stably introduced into Arabidopsis. Histochemical analysis of the transgenic plants showed distinct and specific GUS expression at different stages of anther/pollen development. Transverse sections of flower buds further indicated that the Bc15 promoter activates reporter gene expression in the anther specifically. These findings elucidated the molecular mechanisms of LTP gene regulation during pollen development, and suggested a potential application of the Bc15 promoter in engineering male sterility for hybrid production.

Isolation and Functional Characterization of a Novel Gene Encoding a Dehydration Responsive Element Binding Transcription Factor from Populus euphratica.

Dehydration responsive element binding (DREB) transcription factors (TFs) play a key role in regulating abiotic stress related genes. A new gene (PeDREB2b) encoding an unidentified DRE-binding protein was isolated from 20% PEG6000 treated Populus euphratica Oliv. seedlings by RT-PCR and RACE. Full length of PeDREB2b cDNA was 1110 bp, and an ORF of 870 bp, which encoded 289-amino-acids polypeptide, were included. The deduced amino acid sequence analysis revealed that this protein was a putative AP2/EREBP transcription factor with a conserved AP2/EREBP domain of 64 amino acids and a potential nuclear localization signal (NLS). Based on phylogenetic characterization, PeDREB2b was classified as a member of A-5 group belonged to the DREB family. The PeDREB2b gene is induced by salinization, low temperature, drought and phytohormones GA3, NAA and 6BA, but not by ABA treatment. The fact that the product of PeDREB2b as a DREB transcription factor was verified in our further experiment: the nuclear localization of the gene when it was expressed transiently as a GFP fusion in onion epidermal cells. In addition, PeDREB2b was capable of activating reporter gene expression. To study the salt and drought stress responses for PeDREB2b transgenic Arabidopsis thaliana in detail, integrated physiological, biochemical and genetic approach methods were used. Results indicated that the PeDREB2b gene was over-expressed under stress-inducible rd29A promotor in transgenic plants alleviates the adverse effects of environmental stresses.