High-level Constitutive Expression Research Articles

Targeting Mosquitoes through Generation of an Insecticidal RNAi Yeast Strain Using Cas-CLOVER and Super PiggyBac Engineering in Saccharomyces cerevisiae.

The global deployment of RNAi yeast insecticides involves transitioning from the use of laboratory yeast strains to more robust strains that are suitable for scaled fermentation. In this investigation, the RNA-guided Cas-CLOVER system was used in combination with Piggybac transposase to produce robust Saccharomyces cerevisiae strains with multiple integrated copies of the Sh.463 short hairpin RNA (shRNA) insecticide expression cassette. This enabled the constitutive high-level expression of an insecticidal shRNA corresponding to a target sequence that is conserved in mosquito Shaker genes, but which is not found in non-target organisms. Top-expressing Cas-CLOVER strains performed well in insecticide trials conducted on Aedes, Culex, and Anopheles larvae and adult mosquitoes, which died following consumption of the yeast. Scaled fermentation facilitated the kilogram-scale production of the yeast, which was subsequently heat-killed and dried. These studies indicate that RNAi yeast insecticide production can be scaled, an advancement that may one day facilitate the global distribution of this new mosquito control intervention.

DMD-Associated Dilated Cardiomyopathy: Genotypes, Phenotypes, and Phenocopies.

Variants in the DMD gene, that encodes the cytoskeletal protein, dystrophin, cause a severe form of dilated cardiomyopathy (DCM) associated with high rates of heart failure, heart transplantation, and ventricular arrhythmias. Improved early detection of individuals at risk is needed. Genetic testing of 40 male probands with a potential X-linked genetic cause of primary DCM was undertaken using multi-gene panel sequencing, multiplex polymerase chain reaction, and array comparative genomic hybridization. Variant location was assessed with respect to dystrophin isoform patterns and exon usage. Telomere length was evaluated as a marker of myocardial dysfunction in left ventricular tissue and blood. Four pathogenic/likely pathogenic DMD variants were found in 5 probands (5/40: 12.5%). Only one rare variant was identified by gene panel testing with 3 additional multi-exon deletion/duplications found following targeted assays for structural variants. All of the pathogenic/likely pathogenic DMD variants involved dystrophin exons that had percent spliced-in scores >90, indicating high levels of constitutive expression in the human adult heart. Fifteen DMD variant-negative probands (15/40: 37.5%) had variants in autosomal genes including TTN, BAG3, LMNA, and RBM20. Myocardial telomere length was reduced in patients with DCM irrespective of genotype. No differences in blood telomere length were observed between genotype-positive family members with/without DCM and controls. Primary genetic testing using multi-gene panels has a low yield and specific assays for structural variants are required if DMD-associated cardiomyopathy is suspected. Distinguishing X-linked causes of DCM from autosomal genes that show sex differences in clinical presentation is crucial for informed family management.

Expression strategies for the efficient synthesis of antimicrobial peptides in plastids

Antimicrobial peptides (AMPs) kill microbes or inhibit their growth and are promising next-generation antibiotics. Harnessing their full potential as antimicrobial agents will require methods for cost-effective large-scale production and purification. Here, we explore the possibility to exploit the high protein synthesis capacity of the chloroplast to produce AMPs in plants. Generating a large series of 29 sets of transplastomic tobacco plants expressing nine different AMPs as fusion proteins, we show that high-level constitutive AMP expression results in deleterious plant phenotypes. However, by utilizing inducible expression and fusions to the cleavable carrier protein SUMO, the cytotoxic effects of AMPs and fused AMPs are alleviated and plants with wild-type-like phenotypes are obtained. Importantly, purified AMP fusion proteins display antimicrobial activity independently of proteolytic removal of the carrier. Our work provides expression strategies for the synthesis of toxic polypeptides in chloroplasts, and establishes transplastomic plants as efficient production platform for antimicrobial peptides.

PD-L1 sustains chronic, cancer cell–intrinsic responses to type I interferon, enhancing resistance to DNA damage

Programmed death ligand 1 (PD-L1), an immune-checkpoint protein expressed on cancer cells, also functions independently of the immune system. We found that PD-L1 inhibits the killing of cancer cells in response to DNA damage in an immune-independent manner by suppressing their acute response to type I interferon (IFN; IFN-I). In addition, PD-L1 plays a critical role in sustaining high levels of constitutive expression in cancer cells of a subset of IFN-induced genes, the IFN-related DNA damage resistance signature (IRDS) which, paradoxically, protects cancer cells. The cyclic GMP-AMP synthase-stimulator of the IFN genes (cGAS-STING) pathway is constitutively activated in a subset of cancer cells in the presence of high levels of PD-L1, thus leading to a constitutive, low level of IFN-β expression, which in turn increases IRDS expression. The constitutive low level of IFN-β expression is critical for the survival of cancer cells addicted to self-produced IFN-β. Our study reveals immune-independent functions of PD-L1 that inhibit cytotoxic acute responses to IFN-I and promote protective IRDS expression by supporting protective chronic IFN-I responses, both of which enhance the resistance of cancer cells to DNA damage.

Genome editing of a hybridoma cell line via the CRISPR/Cas9 system: A new approach for constitutive high-level expression of heterologous proteins in eukaryotic system

The power of the CRISPR/Cas9 system has revolutionized genome editing in many fields of biology. These applications have expanded exponentially over recent years, including those regarding protein expression technologies. The CRISPR/Cas9 system avoids random integration of the gene of interest and due to this characteristic can be exploited to obtain a stable cell line for the high-yield expression of recombinant proteins. Here we propose a method to edit a hybridoma cell line for the constitutive expression of proteins of interest using the CRISPR/Cas9 system. First, with the scope of optimizing the method, we replaced part of the light chain of immunoglobulin with the Green Fluorescent Protein (GFP) gene, obtaining a precise knock-in in the hybridoma genome. We confirmed the expression and secretion of GFP into the culture medium via fluorimetric analysis, as well as correct genome editing by RNA sequencing. Then, using the same approach, we included the gene encoding a protein of diagnostic interest, the Bovine Herpesvirus 1 glycoprotein E, in the donor DNA. We obtained a stable clone able to secrete gE protein in fusion with GFP into the culture medium. This result was confirmed by ELISA and Western Blot analysis. This study confirms the suitability of this cell line for the production of proteins of diagnostic interest by stable gene expression in a mammalian system. These experiments will enable the technique to be developed from its proof of concept to more specific applications in the field of infectious disease diagnostics.

Engineering Chloroplasts for High-Level Constitutive or Inducible Transgene Expression.

Expression of transgenes from the plastid genome offers a number of attractions to biotechnologists, with the potential to attain very high protein accumulation levels arguably being the most attractive one. High-level transgene expression is of particular importance in resistance engineering (e.g., for expression of insecticidal proteins) and molecular farming (e.g., for expression of pharmaceutical proteins and industrial enzymes). Over the past decades, the production of many commercially valuable proteins in chloroplast-transgenic (transplastomic) plants has been attempted, including pharmaceutical proteins (e.g., subunit vaccines and protein antibiotics) and industrial enzymes. Although in some cases, spectacularly high foreign protein accumulation levels have been obtained, expression levels were disappointingly poor in other cases. In this review, I summarize our current knowledge about the factors influencing the efficiency of plastid transgene expression, and highlight possible optimization strategies to alleviate problems with poor expression levels. I also discuss available techniques for inducible expression of chloroplast transgenes.

Development and validation of a rapid LC-MS/MS method for the quantification of mycosporines and mycosporine-like amino acids (MAAs) from cyanobacteria

Mycosporines and mycosporine-like amino acids are ultra-violet radiation absorbing compounds produced by lichens, fungi, algae and cyanobacterial species, especially upon exposure to solar ultraviolet radiation. These compounds are photoprotective and some have additional antioxidant functions. Thus, mycosporine-like amino acids may be used as an active ingredient in cosmetic products. Analysis is generally achieved by separation using reverse-phase chromatography and, detection and quantification based upon characteristic absorption spectra and extremely high molar extinction coefficients. Herein, a validated liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) quantification method for mycosporine-like amino acids is presented. Additionally, an easy-to-handle and rapid extraction procedure was developed, which uses only water and volatile additives as the extractor solvents. The LC-MS/MS method was performed with an electrospray ionization source in positive mode using multiple reaction monitoring with a reverse-phase column. The method enabled the accurate determination and quantification of the mycosporine-like amino acids porphyra-334, shinorine and mycosporine-glycine-alanine in a 6 min running time, with detection limits of 0.002 μg mg−1, 0.001 μgmg−1 and 0.005 μg mg−1, respectively. These methods provided a simple extraction and rapid analysis procedure that efficiently identified and quantified these three mycosporine-like amino acids produced by a collection of 29 cyanobacteria. Although more mycosporine-like amino acids have also been identified, the validated method was developed focusing on the most abundant mycosporine-like amino acids produced by the investigated cyanobacteria. Mycosporine-like amino acids could be detected at levels well above the limits of detection for the LC-MS/MS method in 12 strains, irrespective of whether the cultures had been exposed to ultra-violet radiation. High levels of constitutive expression of mycosporine-like amino acids in cyanobacteria has rarely been reported and provides an opportunity to study mechanisms that regulate ultra-violet radiation induced mycosporine-like amino acids biosynthesis in the future.

High-level constitutive expression of leech hyaluronidase with combined strategies in recombinant Pichia pastoris

Hyaluronidases that break down hyaluronan are widely used for preparation of low molecular weight hyaluronan. Leech hyaluronidase (LHyal) is a newly discovered hyaluronidase with outstanding enzymatic properties. The Pichia pastoris expression system of LHyal that depends on AOX1 promoter (PAOX1) has been constructed. However, the addition of the toxic inducer methanol is a big safety concern. Here, a combinational strategy was adopted for constitutive expression of LHyal to high level in P. pastoris. By optimizing the combination of promoters PGAP, PGAP(m), and PTEF1 and signal peptides α-factor, nsB, and sp23, the enzyme activity of extracellular LHyal reached 1.38 × 105U/mL in shake flasks. N-terminal engineering with neutral polar amino acids further increased LHyal activity to 2.06 × 105U/mL. In addition, the impact of overexpressing transcription factors Aft1, Gal4-like, and Yap1 on LHyal production was also investigated. We found the co-expression of Aft1 significantly enhanced the expression of LHyal to 3.03 × 105U/mL. Finally, LHyal activity of 2.12 × 106U/mL was achieved in a 3-L fermenter, with a high productivity of 1.96 × 104U/mL/h. The engineered LHyal-producing Pichia pastoris strains will be more attractive for production of hyaluronidase on industrial scale.

Molecular characterization of a sweet potato stress tolerance-associated trehalose-6-phosphate synthase 1 gene (IbTPS1) in response to abiotic stress

Trehalose-6-phosphate synthase (TPS) plays an important regulatory role in the response of plants to multiple abiotic stresses. However, our knowledge of the stress tolerance functions of TPS genes in sweet potato (Ipomoea batatas [L.] Lam) remains limited. In the present study, we isolated and functionally characterized the sweet potato gene encoding TPS1, IbTPS1. Sequence analysis showed that IbTPS1 belongs to class I TPS proteins and harbors highly conserved acceptor (glucose-6-phosphate [Glc6P]) and donor (uridine diphosphoglucose [UDP-Glc]) binding sites. The IbTPS1 gene showed the highest level of constitutive expression in leaves and storage roots of sweet potato plants and was induced by several environmental stresses including drought, salt, and heat shock. The IbTPS1 protein might be localized to the cytosol. Complementation assay of yeast tps1Δ and tps1Δtps2Δ growth-defective mutants confirmed the TPS activity of IbTPS1, and truncation of the N-terminal extension of IbTPS1 (ΔNIbTPS1) increased the catalytic activity of the protein. Additionally, expression of IbTPS1 in yeast conferred abiotic stress tolerance to dehydration, salinity, and oxidation, and expression of ΔNIbTPS1 further enhanced the tolerance to abiotic stresses. The results of this study advance our understanding of the functions of IbTPS1 under abiotic stress conditions. Our results suggest that the IbTPS1 gene is an excellent candidate for improving the stress tolerance of different crop plants.

Isolation, expression and function analysis of a bZIP transcription factor IbbZIP37 in sweetpotato (Ipomoea batatas L. [Lam

bZIP transcription factor play an important regulatory role in the response to multiple abiotic stresses. However, our knowledge of the stress tolerance functions of bZIP family genes in sweetpotato (Ipomoea batatas [L.] Lam) remains limited. In the present study, we isolated and functionally characterized an IbbZIP37 gene encoding an abiotic stress-inducible bZIP group A transcription factor. Sequence analysis showed that the IbbZIP37 contained a typical bZIP domain and five conserved Ser/Thr kinase phosphorylation sites (RXXS/T). The IbbZIP37 protein was localized in the nucleus and possessed transcriptional activation activity. The results of electrophoretic mobility shift assays indicated that IbbZIP37 can bind to the ABRE cis-element, not do to DRE cis-element in vitro. The IbbZIP37 gene showed the highest level of constitutive expression in root, especially in fibrous root and storage root body. Gene expression was induced by ABA and several environmental stresses including drought, salt and heat shock. Our results suggest that IbbZIP37 is a positive transcription regulator of the abiotic stresses response, which can be used as an excellent candidate for improving the stress tolerance of different crop plants.

The Agrobacterium tumefaciens atu3184 gene, a member of the COG0523 family of GTPases, is regulated by the transcriptional repressor Zur

Analysis of the Agrobacterium tumefaciens C58 genome revealed a potential Zur (zinc uptake regulator) binding site (5′-GATATGTTATTACATTAC-3′, the underlined letters are the center of symmetry of the inverted palindrome) located in the upstream region of atu3184, whose gene product is a member of the COG0523 subfamily of G3E GTPases. The specific interaction of the Zur protein with the 18-bp inverted repeat operator motif in the presence of zinc was demonstrated in vitro by a DNA band shift assay and a DNase I footprinting assay. A LacZ reporter fusion assay further confirmed that Zur negatively regulates atu3184 promoter activity in vivo. The expression of atu3184 was upregulated in response to zinc limitation in the wild-type strain, but the zur mutant strain exhibited high-level constitutive expression of atu3184 under all conditions, irrespective of the zinc levels. It is likely that A. tumefaciens Zur senses zinc and directly regulates the atu3184 promoter by a molecular mechanism similar to that of Escherichia coli Zur, where the operator DNA is surrounded by four Zur monomers forming two dimers bound on the opposite sides of the DNA duplex. Disruption of atu3184 did not affect cell growth under metal-limited conditions and had no effect on the total cellular zinc content. Furthermore, an A. tumefaciens strain lacking atu3184 caused a tumor disease in a host plant.

Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae.

Haploid laboratory strains of Saccharomyces cerevisiae are commonly used for genetic engineering to enable their xylose utilization but little is known about the industrial yeast which is often recognized as diploid and as well as haploid and tetraploid. Here we report three unique signature pathway expression patterns and gene interactions in the centre metabolic pathways that signify xylose utilization of genetically engineered industrial yeast S. cerevisiae NRRL Y-50463, a diploid yeast. Quantitative expression analysis revealed outstanding high levels of constitutive expression of YXI, a synthesized yeast codon-optimized xylose isomerase gene integrated into chromosome XV of strain Y-50463. Comparative expression analysis indicated that the YXI was necessary to initiate the xylose metabolic pathway along with a set of heterologous xylose transporter and utilization facilitating genes including XUT4, XUT6, XKS1 and XYL2. The highly activated transketolase and transaldolase genes TKL1, TKL2, TAL1 and NQM1 as well as their complex interactions in the non-oxidative pentose phosphate pathway branch were critical for the serial of sugar transformation to drive the metabolic flow into glycolysis for increased ethanol production. The significantly increased expression of the entire PRS gene family facilitates functions of the life cycle and biosynthesis superpathway for the yeast. The outstanding higher levels of constitutive expression of YXI and the first insight into the signature pathway expression and the gene interactions in the closely related centre metabolic pathways from the industrial yeast aid continued efforts for development of the next-generation biocatalyst. Our results further suggest the industrial yeast is a desirable delivery vehicle for new strain development for efficient lignocellulose-to-advanced biofuels production.

In vivo selection with lentiviral expression of Bcl2T69A/S70A/S87A mutant in hematopoietic stem cell-transplanted mice.

Current in vivo selections for hematopoietic stem cell (HSC)-based gene therapy are drug dependent and not without risk of cytotoxicity or tumorigenesis. We developed a new in vivo selection system with the non-phosphorylatable Bcl2 mutant Bcl2T69A/S70A/S87A (Bcl2AAA), which makes in vivo selection drug independent and without risk of cytotoxicity or tumorigenesis. We demonstrated in HSC-transplanted mice that Bcl2AAA facilitated efficient in vivo selection in the absence of any exogenously applied drugs under both myeloablative and non-myeloablative conditioning. In mice transplanted with retrovirally transduced sca-1-positive bone marrow cells, the marked cell level increased from 26.38% of input transduced cells to 92.61 ± 0.95% of peripheral blood cells for myeloablative transplantation or to 37.82 ± 6.35% for non-myeloablative transplantation 6 months after transplantation. Bcl2AAA did not induce tumorigenesis and does not influence hematopoiesis and the function of the reconstituted blood system. However, the high-level constitutive expression of Bcl2AAA mediated by retroviral vector induced exhaustion of the marked cells after tertiary transplantation. Fortunately, low-level constitutive expression of Bcl2AAA driven by an internal promoter in lentiviral vector could both maintain the marked cell level (24.13 ± 5.27%, 27.17 ± 5.51%, 24.33 ± 5.08%, and 22.07 ± 4.44% for primary, secondary, tertiary, and quaternary recipients) and avoid the exhaustion of the marked cells even in quaternary recipients. Importantly, the low-level constitutive expression of Bcl2AAA did not induce tumorigenesis. Thus, the in vivo selection employing the low-level constitutive expression of Bcl2AAA provides a general platform which is relevant for widespread applications of gene therapy.

CCL18 – Beyond chemotaxis

The chemokine CCL18 is constitutively expressed in human lung and serum, and is further elevated during pathologic conditions such as allergy, fibrosis and cancer, suggesting that it may participate in both homeostatic and inflammatory processes. Under steady state conditions, CCL18 has chemotactic activity, albeit modest, toward naïve T cells and as such, may be involved in the initiation of the adaptive response. Its chemotactic effect on inflammatory cells is ambiguous as it attracts both regulatory and inflammatory immune cells. CCL18 can also modulate tissue inflammation by inhibiting cell recruitment through binding to glycosaminoglycans with high affinity, thereby displacing other chemokines bound to the endothelial surface. CCL18 induces regulatory phenotype and function of immune cells through direct activation and plays a major role in fibrotic processes, particularly in the lung. Finally, CCL18 is involved in cancer cell activation and migration and also participates in immune tolerance toward cancer. Its high constitutive expression levels and its further up-regulation in many diseases, together with its moderate chemoattractant properties support the fact that this chemokine has activities beyond cell recruitment.

Down regulation of G protein-coupled receptor 137 expression inhibits proliferation and promotes apoptosis in leukemia cells

BackgroundG protein-coupled receptors (GPR) are involved in a wide range of physiological processes, some of which, however, can be hijacked by tumor cells. Over-expression of G protein-coupled receptors 137 (GPR137) are associated with the growth of tumor cells, but under-expression of GPR137 has shown to inhibit cell proliferation in several different types of cancers. Currently, the role of GPR137 in leukemia is still unclear. In this study, the effect of under-expression of GPR137 on inhibiting the proliferation of leukemia cells is explored, to identify a novel target for leukemia treatment.Materials and methodsIn this study, lentivirus-mediated RNA interference (RNAi) was employed to investigate the role of GPR137 in two leukemia cell lines K562 and HL60. The gene expression of GPR137 was analyzed by RT-PCR and its protein expression was determined by Western blot. Flow cytometry and Annexin V/7-AAD Apoptosis Detection Kit was used respectively in cell cycle and apoptosis analysis. The protein expression of CyclinD1, CDK4, BCL-2 and caspase-3 were also determined.ResultsThere was high level of constitutive expression of GPR137 in leukemia cancer cell lines K562 and HL60. Lentivirus-mediated RNAi could significantly down-regulate gene and protein expression of GPR137 in both cell lines. Down regulation of GPR137 was associated with the reduction in proliferation rate and colony forming capacity. In addition, down regulation of GPR137 arrested cells in the G0/G1 phase of cell cycle and induced apoptosis in both leukemia cell lines K562 and HL60.ConclusionsThe expression of GPR137 is associated with the proliferation of leukemia cell lines. Down regulation of GPR137 could inhibit proliferation and promote apoptosis in leukemia cells, which makes it a promising bio-marker and therapeutic target to treat patients with leukemia.

Antisense transcription represses Arabidopsis seed dormancy QTL DOG1 to regulate drought tolerance.

Plants have developed multiple strategies to sense the external environment and to adapt growth accordingly. Delay of germination 1 (DOG1) is a major quantitative trait locus (QTL) for seed dormancy strength in Arabidopsis thaliana that is reported to be expressed exclusively in seeds. DOG1 is extensively regulated, with an antisense transcript (asDOG1) suppressing its expression in seeds. Here, we show that asDOG1 shows high levels in mature plants where it suppresses DOG1 expression under standard growth conditions. Suppression is released by shutting down antisense transcription, which is induced by the plant hormone abscisic acid (ABA) and drought. Loss of asDOG1 results in constitutive high-level DOG1 expression, conferring increased drought tolerance, while inactivation of DOG1 causes enhanced drought sensitivity. The unexpected role of DOG1 in environmental adaptation of mature plants is separate from its function in seed dormancy regulation. The requirement of asDOG1 to respond to ABA and drought demonstrates that antisense transcription is important for sensing and responding to environmental changes in plants.

Phenotype and tissue distribution of CD28H+ immune cell subsets

ABSTRACTCD28H is a newly discovered co-receptor of the human B7 family. CD28H interacts with its ligand B7-H5 and regulates T cell response. Here we showed that CD28H was not expressed on granulocytes, monocytes, myeloid dendritic cells (MDCs), and B cells, but constitutively expressed with moderate levels on memory T cells and with high levels on naïve T cells, innate lymphoid cells (ILCs), natural killer (NK) cells, and plasmacytoid dendritic cells (PDCs) in human peripheral blood. Similar CD28H+ cell profile existed in secondary lymphoid organs and pathological tissues including multiple types of cancers. Further analysis demonstrated that CD28H+ naïve and CD28H+ memory T cells were characterized with increased naïve feature and less effector functional phenotype, respectively. High levels of constitutive CD28H expression on naïve T cells and innate immune cells suggest a potential role of CD28H in innate and adaptive immunity.

Promoter of chrysanthemum actin confers high-level constitutive gene expression in Arabidopsis and chrysanthemum

A promoter that confers high-level constitutive expression of transgenes in crop plants, designated the CmActin promoter, was isolated from chrysanthemum (Chrysanthemum morifolium Ramat. ‘White Wing’). We characterized Arabidopsis and chrysanthemum transformants bearing a CmActin promoter-β-glucuronidase (GUS) reporter construct. Analysis of GUS expression demonstrated that the promoter directed reporter gene expression during all developmental stages and tissues of transgenic Arabidopsis except in seeds. Higher expression of GUS was observed in organs and tissues of transgenic chrysanthemum compared to those of 35S promoter-GUS transgenic plants. In addition, BrSRS expression driven by the CmActin promoter induced dwarf and compact plants with narrow upwardly curled leaves and short petioles. Transgene expression driven by the CmActin promoter was very similar to that of the 35S promoter in Arabidopsis and was significantly stronger than that of the 35S promoter in chrysanthemum, suggesting that the CmActin promoter can induce constitutive gene expression in various plants and that its function is presumably conserved in different species. The CmActin promoter may provide a practical choice for high-level constitutive expression of target genes and could be a useful tool in floriculture plant genetic engineering.

Adaptation to low temperatures in the wild tomato species Solanum chilense.

Molecular adaptation to abiotic stresses in plants is a complex process based mainly on the modifications of gene transcriptional activity and the alteration of protein-protein interactions. We used a combination of population genetic, comparative transcriptomic and plant physiology approaches to investigate the mechanisms of adaptation to low temperatures in Solanum chilense populations distributed along Andean altitudinal gradients. We found that plants from all populations have high chilling tolerance, which does not correlate with temperatures in their native habitats. In contrast, tolerance to freezing shows a significant association with altitude and temperature variables. We also observed the differences in expression patterns of cold-response genes between plants from high- and low-altitude populations. These results suggest that genetic adaptations to low temperatures evolved in high-altitude populations of S.chilense. At the transcriptional level, these adaptations may include high levels of constitutive expression of the genes encoding ICE1, the key transcription factor of the cold signalling pathway, and chloroplast ω-3 fatty acid desaturase FAD7. At the sequence level, a signature of selection associated with the adaptation to high altitudes was detected at the C-terminal part of ICE1 encoding the ACT regulatory domain.

A novel constitutive promoter and its downstream 5′ UTR derived from cotton (Gossypium spp.) drive high-level gene expression in stem and leaf tissues

The development of genetically modified crops requires new promoters and regulatory regions to achieve high gene expression and/or tissue-specific expression patterns in plants. To obtain promoter sequences of plants with new properties, we analyzed the expression traits of the cotton (Gossypium hirsutum) translation elongation factor 1A gene family. The results showed that the GhEF1A8 gene is highly expressed in different organs of cotton plants, and showed much higher transcript levels in stems and leaves. Its promoter (GhEF1A1.7) and the 5′ untranslated region (5′ UTR), comprising a regulatory region named PGhEF1A8, were isolated from cotton and studied in stably transformed tobacco plants. The regulatory region sequences were fused to the β-glucuronidase (GUS) reporter gene to characterize its expression pattern in tobacco. Histochemical and fluorometric GUS activity assays demonstrated that PGhEF1A8 could direct GUS gene expression in all tissues and organs in transgenic tobacco, including leaves, stems, flowers, and roots. The level of GUS activity in the leaves and stems was significantly higher than in cauliflower mosaic virus (CaMV) 35S promoter::GUS plants, but as same as CaMV 35S promoter::GUS plants in flower and root tissues. GUS expression levels decreased 2–10-fold when the 5′ UTR was absent from PGhEF1A8. Deletion analysis of the PGhEF1A8 sequence showed that the region −647 to −323 might possess negative elements that repress transgene expression in tobacco plants. The results suggested that the GhEF1A8 regulation region may represent a practical choice to direct high-level constitutive expression of transgenes and could be a valuable new tool in plant genetic engineering.