Inducible Promoter Research Articles

Production of PHA copolymers consisting of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) by recombinant Halomonas bluephagenesis

Microbial synthesized poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), namely PHBHHx, with different 3HHx monomer ratios have shown biocompatibility, biodegradability and flexibility for commercial uses. This study aims at the low-cost production of PHBHHx with customized and/or rich 3HHx by engineered Halomonas bluephagenesis (H. bluephagenesis), which is a rising-star chassis for Next Generation Industrial Biotechnology (NGIB). Recombinant H. bluephagenesis harboring heterologous PHA synthase (PhaCac) and enoyl coenzyme-A hydratase (PhaJac) could produce PHBHHx from glucose and sodium hexanoate. After rationally fine-tuning the expression levels of phaCac and phaJac employing two inducible promoters, Plac and Plux induced by IPTG and AHL, respectively, the resulting H. bluephagenesis TDC-CJ produced 55 wt% P(3HB-co-14.21 mol% 3HHx). Furthermore, two inducible promoters were replaced by constitutive ones, Pporin68 and Pporin58, respectively, with similar expression strength to remove the additions of inducers. Notably, PHBHHx of high 3HHx ratios, reaching up to 49 mol%, was further obtained by weakening the native β-oxidation pathway. Finally, chromosomally engineered H. bluephagenesis G34 with two expression cassettes of phaCJ on loci G3 and G4 was constructed to achieve the synthesis of P(3HB-co-36 mol% 3HHx) without supplementing antibiotic. For lab-scale fermentation in a 7-L bioreactor, H. bluephagenesis G34 produced PHBHHx with controllable molar fraction of 3HHx from 0-to-37 mol% by designing glucose/hexanoate ratio. These efforts demonstrate that engineering H. bluephagenesis is of great potential for PHBHHx production of low cost for industrial-scale biomanufacturing purpose.

Identification, evolution and expression analyses of the whole genome-wide PEBP gene family in Brassica napus L.

BackgroundWith the release of genomic data for B.rapa, B.oleracea, and B.napus, research on the genetic and molecular functions of Brassica spp. has entered a new stage. PEBP genes in plants play an important role in the transition to flowering as well as seed development and germination. Molecular evolutionary and functional analyses of the PEBP gene family in B.napus based on molecular biology methods can provide a theoretical basis for subsequent investigations of related regulators.ResultsIn this paper, we identified a total of 29 PEBP genes from B.napus that were located on 14 chromosomes and 3 random locations. Most members contained 4 exons and 3 introns; motif 1 and motif 2 were the characteristic motifs of PEBP members. On the basis of intraspecific and interspecific collinearity analyses, it is speculated that fragment replication and genomic replication are the main drivers of for the amplification and evolution of the PEBP gene in the B.napus genome. The results of promoter cis-elements prediction suggest that BnPEBP family genes are inducible promoters, which may directly or indirectly participate in multiple regulatory pathways of plant growth cycle. Furthermore, the tissue-specific expression results show that the expression levels of BnPEBP family genes in different tissues were quite different, but the gene expression organization and patterns of the same subgroup were basically the same. qRT‒PCR revealed certain spatiotemporal patterns in the expression of the PEBP subgroups in roots, stems, leaves, buds, and siliques, was tissue-specific, and related to function.ConclusionsA systematic comparative analysis of the B.napus PEBP gene family was carried out at here. The results of gene identification, phylogenetic tree construction, structural analysis, gene duplication analysis, prediction of promoter cis-elements and interacting proteins, and expression analysis provide a reference for exploring the molecular mechanisms of BnPEBP family genes in future research.

Construction of cell factories for production of patchoulol in Saccharomyces cerevisiae

Patchoulol is an important sesquiterpenoid in the volatile oil of Pogostemon cablin, and is also considered to be the main contributing component to the pharmacological efficacy and fragrance of P. cablin oil, which has antibacterial, antitumor, antioxidant, and other biological activities. Currently, patchoulol and its essential oil blends are in high demand worldwide, but the traditional plant extraction method has many problems such as wasting land and polluting the environment. Therefore, there is an urgent need for a new method to produce patchoulol efficiently and at low cost. To broaden the production method of patchouli and achieve the heterologous production of patchoulol in Saccharomyces cerevisiae, the patchoulol synthase(PS) gene from P. cablin was codon optimized and placed under the inducible strong promoter GAL1 to transfer into the yeast platform strain YTT-T5, thereby obtaining strain PS00 with the production of(4.0±0.3) mg·L~(-1) patchoulol. To improve the conversion rate, this study used protein fusion method to fuse SmFPS gene from Salvia miltiorrhiza with PS gene, leading to increase the yield of patchoulol to(100.9±7.4) mg·L~(-1) by 25-folds. By further optimizing the copy number of the fusion gene, the yield of patchoulol was increased by 90% to(191.1±32.7) mg·L~(-1). By optimizing the fermentation process, the strain was able to achieve a patchouli yield of 2.1 g·L~(-1) in a high-density fermentation system, which was the highest yield so far. This study provides an important basis for the green production of patchoulol.

A robust dual gene ON-OFF toggle directed by two independent promoter-degron pairs.

Switching genes on and off on cue is a cornerstone for understanding gene functions. One contemporary approach for loss-of-function studies of essential genes involves CRISPR-mediated knockout of the endogenous locus in conjunction with the expression of a rescue construct, which can subsequently be turned off to produce a gene inactivation effect in mammalian cell lines. A broadening of this approach would involve simultaneously switching on a second construct to interrogate a gene's functions in the pathway. In this study, we developed a pair of switches that were independently controlled by both inducible promoters and degrons, enabling the toggling between two constructs with comparable kinetics and tightness. The gene-OFF switch was based on TRE transcriptional control coupled with auxin-induced degron-mediated proteolysis. A second independently controlled gene-ON was based on a modified ecdysone promoter and mutated FKBP12-derived destabilization domain degron, allowing acute and tuneable gene activation. This platform facilitates efficient generation of knockout-cell lines containing a two-gene switch that is regulated tightly and can be flipped within a fraction of the time of a cell cycle.

Design of a stable ethanologenic bacterial strain without heterologous plasmids and antibiotic resistance genes for efficient ethanol production from concentrated dairy waste

Engineering sustainable bioprocesses that convert abundant waste into fuels is pivotal for efficient production of renewable energy. We previously engineered an Escherichia coli strain for optimized bioethanol production from lactose-rich wastewater like concentrated whey permeate (CWP), a dairy effluent obtained from whey valorization processes. Although attractive fermentation performances were reached, significant improvements are required to eliminate recombinant plasmids, antibiotic resistances and inducible promoters, and increase ethanol tolerance. Here, we report a new strain with chromosomally integrated ethanologenic pathway under the control of a constitutive promoter, without recombinant plasmids and resistance genes. The strain showed extreme stability in 1-month subculturing, with CWP fermentation performances similar to the ethanologenic plasmid-bearing strain. We then investigated conditions enabling efficient ethanol production and sugar consumption by changing inoculum size and CWP concentration, revealing toxicity- and nutritional-related bottlenecks. The joint increase of ethanol tolerance, via adaptive evolution, and supplementation of small ammonium sulphate amounts (0.05% w/v) enabled a fermentation boost with 6.6% v/v ethanol titer, 1.2 g/L/h rate, 82.5% yield, and cell viability increased by three orders of magnitude. Our strain has attractive features for industrial settings and represents a relevant improvement in the existing ethanol production biotechnologies.

PYHIN protein IFI207 regulates cytokine transcription and IRF7 and contributes to the establishment of K. pneumoniae infection

PYHIN proteins AIM2 and IFI204 sense pathogen DNA, while other PYHINs have been shown to regulate host gene expression through as-yet unclear mechanisms. We characterize mouse PYHIN IFI207, which we find is not involved in DNA sensing but rather is required for cytokine promoter induction in macrophages. IFI207 co-localizes with both active RNA polymerase II (RNA Pol II) and IRF7 in the nucleus and enhances IRF7-dependent gene promoter induction. Generation of Ifi207-/- mice shows no role for IFI207 in autoimmunity. Rather, IFI207 is required for the establishment of a Klebsiella pneumoniae lung infection and for Klebsiella macrophage phagocytosis. These insights into IFI207 function illustrate that PYHINs can have distinct roles in innate immunity independent of DNA sensing and highlight the need to better characterize the whole mouse locus, one gene at a time.

Identification of a Far-Red Light-Inducible Promoter that Exhibits Light Intensity Dependency and Reversibility in a Cyanobacterium.

As the demand for sustainable energy has increased, photoautotrophic cyanobacteria have become a popular platform for developing tools in synthetic biology. Although genetic tools are generally available for several model cyanobacteria, such tools have not yet been developed for many other strains potentially suitable for industrial applications. Additionally, most inducible promoters in cyanobacteria are controlled by chemical compounds, but adding chemicals into growth media on an industrial scale is neither cost-effective nor environmentally friendly. Although using light-controlled promoters is an alternative approach, only a cyanobacterial expression system inducible by green light has so far been described and employed for such applications. In this study, we have established a conjugation-based technique to express a reporter gene (eyfp) in the nonmodel cyanobacterium, Chlorogloeopsis fritschii PCC 9212. We also identified a promoter specifically activated by far-red light from the Far-Red Light Photoacclimation gene cluster of Leptolyngbya sp. JSC-1. This promoter, PchlFJSC1, was successfully used to drive eyfp expression. PchlFJSC1 is tightly regulated by light quality (i.e., wavelength) and leads to an approximately 30-fold increase in EYFP production when cells were exposed to far-red light. The induction level was controlled by the far-red light intensity, and induction stopped when cells were returned to visible light. This system has the potential for further applications in cyanobacteria by providing an additional choice of light wavelength to control gene expression. Collectively, this study developed a functional gene-expression system for C. fritschii PCC 9212 that can be regulated by exposing cells to far-red light.

OsAMT1.1 Expression by Nitrate-Inducible Promoter of OsNAR2.1 Increases Nitrogen Use Efficiency and Rice Yield

Nitrate (NO3–) and ammonium (NH4+) are two main inorganic nitrogen (N) sources during crop growth. Here, we enhanced the expression of OsAMT1.1, which encodes a NH4+ transporter, using the NO3–-inducible promoter of OsNAR2.1 and an ubiquitin promoter in transgenic rice plants. Under field condition of 120 kg /hm2 N, agronomic N use efficiency, N recovery efficiency and N transport efficiency, and grain yield of the pOsNAR2.1:OsAMT1.1 transgenic lines were increased compared with those of the wild type (WT) and the pUbi:OsAMT1.1 transgenic plants. Under 2.0 mmol/L NO3– + 0.5 mmol/L NH4+ and 0.5 mmol/L NO3– + 2.0 mmol/L NH4+ conditions of hydroponic culture, compared with the WT, both biomass and total N content were increased in the pOsNAR2.1:OsAMT1.1 transgenic lines. However, biomass was significantly reduced in pUbi:OsAMT1.1 transgenic plants under 0.5 mmol/L NO3– + 2.0 mmol/L NH4+ condition. The lines expressing pOsNAR2.1:OsAMT1.1 exhibited increased OsAMT1.1 expression and 15NH4+ influx in roots under both 2.0 mmol/L NO3– + 0.5 mmol/L NH4+ and 0.5 mmol/L NO3– + 2.0 mmol/L NH4+ conditions. Our study showed that expression of OsAMT1.1 can be promoted when driven by the OsNAR2.1 promoter, especially under high-level nitrate condition, leading to enhancement of NH4+ uptake, N use efficiency and grain yield.

Inducible Synthetic Growth Regulation Using the ClpXP Proteasome Enhances cis,cis-Muconic Acid and Glycolic Acid Yields in Saccharomyces cerevisiae.

Engineered microbial cells can produce sustainable chemistry, but the production competes for resources with growth. Inducible synthetic control over the resource use would enable fast accumulation of sufficient biomass and then divert the resources to production. We developed inducible synthetic resource-use control overSaccharomyces cerevisiae by expressing a bacterial ClpXP proteasome from an inducible promoter. By individually targeting growth-essential metabolic enzymes Aro1, Hom3, and Acc1 to the ClpXP proteasome, cell growth could be efficiently repressed during cultivation. The ClpXP proteasome was specific to the target proteins, and there was no reduction in the targets when ClpXP was not induced. The inducible growth repression improved product yields from glucose (cis,cis-muconic acid) and per biomass (cis,cis-muconic acid and glycolic acid). The inducible ClpXP proteasome tackles uncertainties in strain optimization by enabling model-guided repression of competing, growth-essential, and metabolic enzymes. Most importantly, it allows improving production without compromising biomass accumulation when uninduced; therefore, it is expected to mitigate strain stability and low productivity challenges.

A self‐propagating, barcoded transposon system for the dynamic rewiring of genomic networks

In bacteria, natural transposon mobilization can drive adaptive genomic rearrangements. Here, we build on this capability and develop an inducible, self‐propagating transposon platform for continuous genome‐wide mutagenesis and the dynamic rewiring of gene networks in bacteria. We first use the platform to study the impact of transposon functionalization on the evolution of parallel Escherichia coli populations toward diverse carbon source utilization and antibiotic resistance phenotypes. We then develop a modular, combinatorial assembly pipeline for the functionalization of transposons with synthetic or endogenous gene regulatory elements (e.g., inducible promoters) as well as DNA barcodes. We compare parallel evolutions across alternating carbon sources and demonstrate the emergence of inducible, multigenic phenotypes and the ease with which barcoded transposons can be tracked longitudinally to identify the causative rewiring of gene networks. This work establishes a synthetic transposon platform that can be used to optimize strains for industrial and therapeutic applications, for example, by rewiring gene networks to improve growth on diverse feedstocks, as well as help address fundamental questions about the dynamic processes that have sculpted extant gene networks.

Enabling growth-decoupled Komagataella phaffii recombinant protein production based on the methanol-free PDH promoter.

The current transition towards the circular bioeconomy requires a rational development of biorefineries to sustainably fulfill the present demands. The use of Komagataella phaffii (Pichia pastoris) can meet this challenge, since it has the capability to use crude glycerol as a carbon-source, a by-product from the biodiesel industry, while producing high- and low-added value products. Recombinant protein production (RPP) using K. phaffii has often been driven either by the methanol induced AOX1 promoter (P AOX1 ) and/or the constitutive GAP promoter (P GAP ). In the last years, strong efforts have been focused on developing novel expression systems that expand the toolbox variety of K. phaffii to efficiently produce diverse proteins that requires different strategies. In this work, a study was conducted towards the development of methanol-free expression system based on a heat-shock gene promoter (PDH) using glycerol as sole carbon source. Using this promoter, the recombinant expression is strongly induced in carbon-starving conditions. The classical P GAP was used as a benchmark, taking for both strains the lipase B from Candida antarctica (CalB) as model protein. Titer of CalB expressed under PDH outperformed P GAP controlled expression in shake-flask cultivations when using a slow-release continuous feeding technology, confirming that PDH is induced under pseudo-starving conditions. This increase was also confirmed in fed-batch cultivations. Several optimization rounds were carried out for PDH under different feeding and osmolarity conditions. In all of them the PDH controlled process outperformed the P GAP one in regard to CalB titer. The best PDH approach reached 3.6-fold more specific productivity than P GAP fed-batch at low μ. Compared to the optimum approach for P GAP -based process, the best PDH fed-batch strategy resulted in 2.3-fold higher titer, while the specific productivity was very similar. To summarize, PDH is an inducible promoter that exhibited a non-coupled growth regulation showing high performance, which provides a methanol-free additional solution to the usual growth-coupled systems for RPP. Thus, this novel system emerges as a potential alternative for K. phaffii RPP bioprocess and for revaluing crude glycerol, promoting the transition towards a circular economy.

Soluble amyloid-β dimers are resistant to amyloid-β prion conversion in vivo suggesting antiprion properties.

According to the instructions for authors, short communications should be published without abstract.

Ectopic expression of pigeonpea Orf147 gene imparts partial sterility in Cicer arietinum

Orf147, a cytotoxic peptide, has been found to cause cytoplasmic male sterility (CMS) in Cajanus cajanifolius (pigeonpea). In our study, Orf147 was introduced into self-pollinating Cicer arietinum (chickpea) using Agrobacterium-mediated transformation for induction of CMS. The stable integration and expression of the transgene has been assessed through PCR and qRT-PCR analysis. In addition, phenotypic sterility analysis has been performed, considering developmental parameters like flower development, pod formation and flower drop. Transgene inheritance analysis demonstrates that out of the five PCR positive events in the T0 generation, two events have segregated according to the Mendelian segregation ratio (3:1) in the T2 generation. Further, pollen viability test using microscopic analysis confirms the induction of partial CMS in transgenic chickpea. The study holds significant value regarding the heterosis of self-pollinating legumes like chickpea. As a part of the prospect, exploring inducible promoters of species-specific or related legumes would be the next step to developing a two-line hybrid system.

Kisspeptin induces Kiss-1 and GnRH gene expression in mHypoA-55 hypothalamic cell models: Involvement of the ERK and PKA signaling pathways

mHypoA-55 cells are kisspeptin-expressing neuronal cells originating from the arcuate nucleus of the mouse hypothalamus. These cells are called KNDy neurons because they co-express kisspeptin, neurokinin B, and dynorphin A. In addition, they express gonadotropin-releasing hormone (GnRH).Here, we found that kisspeptin 10 (KP10) increased Kiss-1 (encoding kisspeptin) and GnRH gene expression in kisspeptin receptor (Kiss-1R)-overexpressing mHypoA-55 cells. KP10 greatly increased serum response element (SRE) promoter activity, which is a target of extracellular signal-regulated kinase (ERK) (20.0 ± 2.54-fold). KP10 also increased cAMP-response element (CRE) promoter activity in these cells (2.32 ± 0.36-fold). KP10-increased SRE promoter activity was significantly prevented in the presence of PD098095, a MEK kinase (MEKK) inhibitor, and KP10-induced CRE promoter activity was also inhibited by PD098059. Similarly, H89, a protein kinase A (PKA) inhibitor, significantly inhibited the KP10 induction of SRE and CRE promoters. KP10-induced Kiss-1 and GnRH gene expressions were inhibited in the presence of PD098059. Likewise, H89 significantly inhibited the KP10-induced increase in Kiss-1 and GnRH. Transfection of mHypoA-55 cells with constitutively active MEKK (pFC-MEKK) increased SRE and CRE promoter activities by 9.75 ± 1.77- and 1.36 ± 0.12-fold, respectively. Induction of constitutively active PKA (pFC-PKA) also increased SRE and CRE promoter activities by 2.41 ± 0.42- and 40.71 ± 7.77-fold, respectively. Furthermore, pFC-MEKK and -PKA transfection of mHypoA-55 cells increased both Kiss-1 and GnRH gene expression.Our current observations suggest that KP10 increases both the ERK and PKA pathways and that both pathways mutually interact in mHypoA-55 hypothalamic cells. Activation of both ERK and PKA signaling might be necessary to induce Kiss-1 and GnRH gene expressions.

Design of Lactococcus lactis Strains Producing Garvicin A and/or Garvicin Q, Either Alone or Together with Nisin A or Nisin Z and High Antimicrobial Activity against Lactococcus garvieae

Lactococcus garvieae is a main ichthyopathogen in rainbow trout (Oncorhynchus mykiss, Walbaum) farming, although bacteriocinogenic L. garvieae with antimicrobial activity against virulent strains of this species have also been identified. Some of the bacteriocins characterized, such as garvicin A (GarA) and garvicin Q (GarQ), may show potential for the control of the virulent L. garvieae in food, feed and other biotechnological applications. In this study, we report on the design of Lactococcus lactis strains that produce the bacteriocins GarA and/or GarQ, either alone or together with nisin A (NisA) or nisin Z (NisZ). Synthetic genes encoding the signal peptide of the lactococcal protein Usp45 (SPusp45), fused to mature GarA (lgnA) and/or mature GarQ (garQ) and their associated immunity genes (lgnI and garI, respectively), were cloned into the protein expression vectors pMG36c, which contains the P32 constitutive promoter, and pNZ8048c, which contains the inducible PnisA promoter. The transformation of recombinant vectors into lactococcal cells allowed for the production of GarA and/or GarQ by L. lactis subsp. cremoris NZ9000 and their co-production with NisA by Lactococcus lactis subsp. lactis DPC5598 and L. lactis subsp. lactis BB24. The strains L. lactis subsp. cremoris WA2-67 (pJFQI), a producer of GarQ and NisZ, and L. lactis subsp. cremoris WA2-67 (pJFQIAI), a producer of GarA, GarQ and NisZ, demonstrated the highest antimicrobial activity (5.1- to 10.7-fold and 17.3- to 68.2-fold, respectively) against virulent L. garvieae strains.

Development of a cellular model to study L-DOPA decarboxylase deregulation in the pathogenesis of schizophrenia

IntroductionSchizophrenia (SZ) is an inherited mental illness that affects 1% of the world’s population. Schizophrenia is a common functional psychosis with some unifying features that appears to have a universal distribution. Much of the research on SZ seeks to identify physiological, biochemical, or genetic features that differ between patients and healthy individuals. Biochemical factors represent an imbalance of certain biochemical substances in the brain, especially neurotransmitters. Early studies focused on the brain biochemistry of patients with SZ in terms of dysregulation of the neurotransmitter network. There is evidence that elevated dopamine concentrations are associated with positive symptoms (i.e., hallucinations, delirium) of the disease. For example, L-DOPA decarboxylase (DDC) is an enzyme involved directly in dopamine and serotonin synthesis and indirectly in noradrenaline synthesis. Therefore, the DDC gene can be considered a candidate gene for schizophrenia and its activity is a good candidate for functional analysis via epigenetic repression.ObjectivesWe aimed to create a cellular model with a deregulated DDC gene.MethodsWe constructed two lentiviral vectors, one expressing the dCas9-KRAB-MeCP2 repressor under the control of a synthetic tetracycline inducible promoter, and the other carrying a cassette expressing two sgRNAs with spacers against the DDC promoter. The SH-SY5Y cell line was sequentially stably transduced with both lentiviral constructs, and cells carrying both constructs were selected by the fluorescence of the GFP and RFP reporter proteins encoded in the lentiviral construct backbones.ResultsMethyl-sensitive real-time PCR followed by high-resolution fusion showed methylation of the DDC promoter. Correspondingly, real-time PCR showed a two-fold decrease in DDC expression in SH-SY5Y during tetracycline-induced expression of the CRISPR repressor.ConclusionsWe developed a cellular model to study the contribution of DDC deregulation to SZ-related molecular mechanisms.Disclosure of InterestNone Declared

Engineering of a probiotic yeast for the production and secretion of medium-chain fatty acids antagonistic to an opportunistic pathogen Candida albicans.

Candida albicans is an opportunistic pathogen, with its infection as one of the causes of morbidity or mortality. Notably, the probiotic yeast Saccharomyces cerevisiae var. boulardii has shown the potential to fight against Candida infections. In this study, we aimed to engineer a commercial boulardii strain to produce medium-chain fatty acids (MCFAs) with antagonistic effects against C. albicans. First, we identified and characterized a boulardii strain and created its auxotrophic strain Δura3. Next, we constructed and expressed a heterologous MCFA biosynthetic pathway under the control of inducible and constitutive promoters. Aside from examining MCFA production and secretion, we confirmed MCFAs' effects on C. albicans' anti-biofilm and anti-hyphal formations and the immunomodulatory effect of MCFA-containing supernatants on Caco-2 cells. We found that under constitutive promoters, the engineered boulardii strain constitutively produced and secreted a mixture of C6:0, C8:0, and C10:0. The secreted MCFAs then reduced biofilm and hyphal formations in C. albicans SC5314. We also confirmed that MCFAs upregulated the expression of virulence-related genes in SC5314. Furthermore, we found that the constitutively produced MCFAs in the supernatant induced the upregulation of immune response genes in Caco-2 cells co-cultured with SC5314, indicating MCFAs' roles in immunomodulation. Overall, the engineered boulardii strain produced and secreted MCFAs, as well as demonstrated antagonistic effects against C. albicans SC5314 and immune-modulatory effects in Caco-2. To our knowledge, this represents the first study tackling the metabolic engineering of a commercial probiotic yeast strain to constitutively produce and secrete MCFAs showing anti-Candida effects. Our study forms the basis of the potential development of a live biotherapeutics probiotic yeast against Candida infections through metabolic engineering strategies.

Scarless DNA Recombineering.

The method described here allows editing of the bacterial genome without leaving any secondary changes (scars) behind. This method uses a tripartite selectable and counterselectable cassette comprising an antibiotic-resistance gene (cat or kan) and the tetR repressor gene linked to a Ptet promoter-ccdB toxin gene fusion. In the absence of induction, the tetR gene product represses the Ptet promoter, preventing ccdB expression. The cassette is first inserted at the target site by selecting for chloramphenicol or kanamycin resistance. It is subsequently replaced by the sequence of interest by selecting for growth in the presence of anhydrotetracycline (AHTc), which inactivates the TetR repressor thereby causing CcdB-induced lethality. Unlike other CcdB-based counterselection schemes, which require specifically designed λ-Red delivery plasmids, the system described here uses the popular plasmid pKD46 as the source of λ-Red functions. This protocol allows a wide variety of modifications, including the intragenic insertion of fluorescent or epitope tags, gene replacements, deletions, and single base-pair substitutions, to be made. In addition, the procedure can be used to place the inducible Ptet promoter at a chosen position in the bacterial chromosome.

Novel inducible promoter DREB1G cloned from date palm exhibits high fold expression over AtRD29 to drought and salinity stress

Right and timely expression of the stress regulatory genes is required for plants to compete against abiotic stresses; it necessitates the isolation and characterization of stress-responsive promoters for improving crops' tolerance to abiotic stresses. Dehydration Responsive Element Binding (DREB) regulates the expression of numerous stress-responsive genes in plants and leads an inevitable role in the adaptation of plants to abiotic stresses. In this study, the promoter region of Phoenix dactylifera (Date palm, a major fruit crop of the arid region) PdDREB1G gene was isolated and characterized for the first time. A comparison of the activity of two promoter fragments, 880 bp (DS) and 1.6 kb (DF) of PdDREB1G to AtRD29A was performed. Histochemical assay displayed remarkable GUS staining and RT-qPCR analysis confirmed the induction of GUS expression in T3 plants of transformed tobacco subjected to different abiotic stresses. Furthermore, compared with the widely used AtRD29A promoter, the relative expression of GUS in leaves by DS and DF was three and twofold higher under salt stress, respectively, while it was twofold in polyethylene glycol (PEG) and abscisic acid (ABA) for DS. Under SA stress, DF and DS displayed 1.5 and onefold expression in leaves, respectively. In the root, DS showed a fourfold increased expression in salt, threefold in PEG and ABA, and twofold in SA. Hence, the DS promoter characterized in the present study becomes a choice over RD29A for abiotic stress responses and is useful to develop stress-tolerant transgenic plants by inducing the expression of stress-inducible genes on stress.

Molecular cloning and functional characterization of the promoter of a novel Aspergillus flavus inducible gene (AhOMT1) from peanut.

Peanut is an important oil and food legume crop grown in more than one hundred countries, but the yield and quality are often impaired by different pathogens and diseases, especially aflatoxins jeopardizing human health and causing global concerns. For better management of aflatoxin contamination, we report the cloning and characterization of a novel A. flavus inducible promoter of the O-methyltransferase gene (AhOMT1) from peanut. The AhOMT1 gene was identified as the highest inducible gene by A. flavus infection through genome-wide microarray analysis and verified by qRT-PCR analysis. AhOMT1 gene was studied in detail, and its promoter, fussed with the GUS gene, was introduced into Arabidopsis to generate homozygous transgenic lines. Expression of GUS gene was studied in transgenic plants under the infection of A. flavus. The analysis of AhOMT1 gene characterized by in silico assay, RNAseq, and qRT-PCR revealed minute expression in different organs and tissues with trace or no response to low temperature, drought, hormones, Ca2+, and bacterial stresses, but highly induced by A. flavus infection. It contains four exons encoding 297 aa predicted to transfer the methyl group of S-adenosyl-L-methionine (SAM). The promoter contains different cis-elements responsible for its expression characteristics. Functional characterization of AhOMT1P in transgenic Arabidopsis plants demonstrated highly inducible behavior only under A. flavus infection. The transgenic plants did not show GUS expression in any tissue(s) without inoculation of A. flavus spores. However, GUS activity increased significantly after inoculation of A. flavus and maintained a high level of expression after 48 hours of infection. These results provided a novel way for future management of peanut aflatoxins contamination through driving resistance genes in A. flavus inducible manner.