scholarly journals A Synthetic Strong and Constitutive Promoter Derived from the Stellaria media pro-SmAMP1 and pro-SmAMP2 Promoters for Effective Transgene Expression in Plants

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1407
Author(s):  
Larisa N. Efremova ◽  
Svetlana R. Strelnikova ◽  
Guzel R. Gazizova ◽  
Elena A. Minkina ◽  
Roman A. Komakhin
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Core Promoter ◽  
Crop Improvement ◽  
Negative Regulator ◽  
Stellaria Media ◽  
Synthetic Promoters ◽  
Cis Element ◽  
Transgenic Cells ◽  
Selection Of

Synthetic promoters are vital for genetic engineering-based strategies for crop improvement, but effective methodologies for their creation and systematic testing are lacking. We report here on the comparative analysis of the promoters pro-SmAMP1 and pro-SmAMP2 from Stellaria media ANTIMICROBIAL PEPTIDE1 (AMP1) and ANTIMICROBIAL PEPTIDE2 (AMP2). These promoters are more effective than the well-known Cauliflower mosaic virus 35S promoter. Although these promoters share about 94% identity, the pro-SmAMP1 promoter demonstrated stronger transient expression of a reporter gene in Agrobacterium infiltration of Nicotiana benthamiana leaves, while the pro-SmAMP2 promoter was more effective for the selection of transgenic tobacco (Nicotiana tabacum) cells when driving a selectable marker. Using the cap analysis of gene expression method, we detected no differences in the structure of the transcription start sites for either promoter in transgenic plants. For both promoters, we used fine-scale deletion analysis to identify 160 bp-long sequences that retain the unique properties of each promoter. With the use of chimeric promoters and directed mutagenesis, we demonstrated that the superiority of the pro-SmAMP1 promoter for Agrobacterium-mediated infiltration is caused by the proline-inducible ACTCAT cis-element strictly positioned relative to the TATA box in the core promoter. Surprisingly, the ACTCAT cis-element not only activated but also suppressed the efficiency of the pro-SmAMP1 promoter under proline stress. The absence of the ACTCAT cis-element and CAANNNNATC motif (negative regulator) in the pro-SmAMP2 promoter provided a more constitutive gene expression profile and better selection of transgenic cells on selective medium. We created a new synthetic promoter that enjoys high effectiveness both in transient expression and in selection of transgenic cells. Intact promoters with differing properties and high degrees of sequence identity may thus be used as a basis for the creation of new synthetic promoters for precise and coordinated gene expression.

scholarly journals Large-scale analysis of Drosophila core promoter function using synthetic promoters

2020 ◽  
Author(s):  
Zhan Qi ◽  
Christophe Jung ◽  
Peter Bandilla ◽  
Claudia Ludwig ◽  
Mark Heron ◽  
...  
Keyword(s):  
Gene Expression ◽  
Large Scale ◽  
Function Analysis ◽  
Core Promoter ◽  
Luciferase Reporter ◽  
Sequence Motifs ◽  
Promoter Function ◽  
Synthetic Promoters ◽  
The Individual ◽  
The Impact

SUMMARYThe core promoter, the region immediately surrounding the transcription start site, plays a central role in setting metazoan gene expression levels, but how exactly it ‘computes’ expression remains poorly understood. To dissect core promoter function, we carried out a comprehensive structure-function analysis to measure synthetic promoters’ activities, with and without an external stimulus (hormonal activation). By using robotics and a dual-luciferase reporter assay, we tested ∼3000 mutational variants representing 19 different Drosophila melanogaster promoter architectures. We explored the impact of different types of mutations, including knockout of individual sequence motifs and motif combinations, variations of motif strength, positioning, and flanking sequences. We observe strong effects of the mutations on activity, and a linear combination of the individual motif features can largely account for the combinatorial effects on core promoter activity. Our findings shed new light on the quantitative assessment of gene expression, a fundamental process in all metazoans.

scholarly journals Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach

2011 ◽  
Vol 77 (22) ◽  
pp. 7905-7914 ◽  
Author(s):  
John Blazeck ◽  
Leqian Liu ◽  
Heidi Redden ◽  
Hal Alper
Keyword(s):  
Gene Expression ◽  
Yarrowia Lipolytica ◽  
Core Promoter ◽  
Pathway Engineering ◽  
Mrna Levels ◽  
Enhancer Element ◽  
Content Type ◽  
Synthetic Promoters ◽  
Native Promoter ◽  
Hybrid Promoter

ABSTRACTThe development of strong and tunable promoter elements is necessary to enable metabolic and pathway engineering applications for any host organism. Here, we have expanded and generalized a hybrid promoter approach to produce libraries of high-expressing, tunable promoters in the nonconventional yeastYarrowia lipolytica. These synthetic promoters are comprised of two modular components: the enhancer element and the core promoter element. By exploiting this basic promoter architecture, we have overcome native expression limitations and provided a strategy for both increasing the native promoter capacity and producing libraries for tunable gene expression in a cellular system with ill-defined genetic tools. In doing so, this work has created the strongest promoters ever reported forY. lipolytica. Furthermore, we have characterized these promoters at the single-cell level through the use of a developed fluorescence-based assay as well as at the transcriptional and whole-cell levels. The resulting promoter libraries exhibited a range of more than 400-fold in terms of mRNA levels, and the strongest promoters in this set had 8-fold-higher fluorescence levels than those of typically used endogenous promoters. These results suggest that promoters inY. lipolyticaare enhancer limited and that this limitation can be partially or fully alleviated through the addition of tandem copies of upstream activation sequences (UASs). Finally, this work illustrates that tandem copies of UAS regions can serve as synthetic transcriptional amplifiers that may be generically used to increase the expression levels of promoters.

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

scholarly journals Insight into the bZIP Gene Family in Solanum tuberosum: Genome and Transcriptome Analysis to Understand the Roles of Gene Diversification in Spatiotemporal Gene Expression and Function

2020 ◽  
Vol 22 (1) ◽  
pp. 253
Author(s):  
Venura Herath ◽  
Jeanmarie Verchot
Keyword(s):  
Gene Expression ◽  
Gene Family ◽  
Functional Groups ◽  
Leucine Zipper ◽  
Expression Profiles ◽  
Crop Improvement ◽  
Potato Virus X ◽  
Tissue Growth ◽  
Abiotic Stress Response ◽  
Sequence Alignments

The basic region-leucine zipper (bZIP) transcription factors (TFs) form homodimers and heterodimers via the coil–coil region. The bZIP dimerization network influences gene expression across plant development and in response to a range of environmental stresses. The recent release of the most comprehensive potato reference genome was used to identify 80 StbZIP genes and to characterize their gene structure, phylogenetic relationships, and gene expression profiles. The StbZIP genes have undergone 22 segmental and one tandem duplication events. Ka/Ks analysis suggested that most duplications experienced purifying selection. Amino acid sequence alignments and phylogenetic comparisons made with the Arabidopsis bZIP family were used to assign the StbZIP genes to functional groups based on the Arabidopsis orthologs. The patterns of introns and exons were conserved within the assigned functional groups which are supportive of the phylogeny and evidence of a common progenitor. Inspection of the leucine repeat heptads within the bZIP domains identified a pattern of attractive pairs favoring homodimerization, and repulsive pairs favoring heterodimerization. These patterns of attractive and repulsive heptads were similar within each functional group for Arabidopsis and S. tuberosum orthologs. High-throughput RNA-seq data indicated the most highly expressed and repressed genes that might play significant roles in tissue growth and development, abiotic stress response, and response to pathogens including Potato virus X. These data provide useful information for further functional analysis of the StbZIP gene family and their potential applications in crop improvement.

scholarly journals Spatio-temporal selection of reference genes in the two congeneric species of Glycyrrhiza

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuping Li ◽  
Xiaoju Liang ◽  
Xuguo Zhou ◽  
Yu An ◽  
Ming Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Developmental Stage ◽  
Reference Genes ◽  
Developmental Stages ◽  
Individual Factors ◽  
Congeneric Species ◽  
Spatio Temporal ◽  
The Stability ◽  
Different Tissues ◽  
Selection Of

AbstractGlycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal (developmental stage) interactions (CAC, R3HDM2 and DNAJ). Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.

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