Rational design and testing of abiotic stress‐inducible synthetic promoters from poplar cis ‐regulatory elements

Abstract Promoters serve a critical role in establishing baseline transcriptional capacity through the recruitment of proteins, including transcription factors. Previously, a paucity of data for cis-regulatory elements in plants meant that it was challenging to determine which sequence elements in plant promoter sequences contributed to transcriptional function. In this study, we have identified functional elements in the promoters of plant genes and plant pathogens that utilize plant transcriptional machinery for gene expression. We have established a quantitative experimental system to investigate transcriptional function, investigating how identity, density and position contribute to regulatory function. We then identified permissive architectures for minimal synthetic plant promoters enabling the computational design of a suite of synthetic promoters of different strengths. These have been used to regulate the relative expression of output genes in simple genetic devices.

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Rational Design of Minimal Synthetic Promoters for Plants

10.1101/2020.05.14.095406 ◽

2020 ◽

Author(s):

Yaomin Cai ◽

Kalyani Kallam ◽

Henry Tidd ◽

Giovanni Gendarini ◽

Amanda Salzman ◽

...

Keyword(s):

Rational Design ◽

Plant Pathogens ◽

Critical Role ◽

Experimental System ◽

Computational Design ◽

Regulatory Elements ◽

Regulatory Function ◽

Synthetic Promoters ◽

Sequence Elements ◽

Plant Promoters

ABSTRACTPromoters serve a critical role in establishing baseline transcriptional capacity through the recruitment of proteins, including transcription factors (TFs). Previously, a paucity of data for cis-regulatory elements in plants meant that it was challenging to determine which sequence elements in plant promoter sequences contributed to transcriptional function. In this study, we have identified functional elements in the promoters of plant genes and plant pathogens that utilise plant transcriptional machinery for gene expression. We have established a quantitative experimental system to investigate transcriptional function, investigating how identity, density and position contribute to regulatory function. We then identified permissive architectures for minimal synthetic plant promoters enabling computational design of a suite of synthetic promoters of different strengths. These have been used to regulate the relative expression of output genes in simple genetic devices.

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Genome-wide identification and expression analysis of Raffinose synthetase family in cotton

BMC Bioinformatics ◽

10.1186/s12859-021-04276-4 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Ruifeng Cui ◽

Xiaoge Wang ◽

Waqar Afzal Malik ◽

Xuke Lu ◽

Xiugui Chen ◽

...

Keyword(s):

Abiotic Stress ◽

Expression Patterns ◽

Regulatory Elements ◽

Plant Leaves ◽

Motif Analysis ◽

Expression Levels ◽

Genome Wide ◽

Salt And Drought Stress ◽

Key Genes ◽

Stress Gene

Abstract Background The Raffinose synthetase (RAFS) genes superfamily is critical for the synthesis of raffinose, which accumulates in plant leaves under abiotic stress. However, it remains unclear whether RAFS contributes to resistance to abiotic stress in plants, specifically in the Gossypium species. Results In this study, we identified 74 RAFS genes from G. hirsutum, G. barbadense, G. arboreum and G. raimondii by using a series of bioinformatic methods. Phylogenetic analysis showed that the RAFS gene family in the four Gossypium species could be divided into four major clades; the relatively uniform distribution of the gene number in each species ranged from 12 to 25 based on species ploidy, most likely resulting from an ancient whole-genome polyploidization. Gene motif analysis showed that the RAFS gene structure was relatively conservative. Promoter analysis for cis-regulatory elements showed that some RAFS genes might be regulated by gibberellins and abscisic acid, which might influence their expression levels. Moreover, we further examined the functions of RAFS under cold, heat, salt and drought stress conditions, based on the expression profile and co-expression network of RAFS genes in Gossypium species. Transcriptome analysis suggested that RAFS genes in clade III are highly expressed in organs such as seed, root, cotyledon, ovule and fiber, and under abiotic stress in particular, indicating the involvement of genes belonging to clade III in resistance to abiotic stress. Gene co-expressed network analysis showed that GhRFS2A-GhRFS6A, GhRFS6D, GhRFS7D and GhRFS8A-GhRFS11A were key genes, with high expression levels under salt, drought, cold and heat stress. Conclusion The findings may provide insights into the evolutionary relationships and expression patterns of RAFS genes in Gossypium species and a theoretical basis for the identification of stress resistance materials in cotton.

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Genome-wide promoter analysis, homology modeling and protein interaction network of Dehydration Responsive Element Binding (DREB) gene family in Solanum tuberosum

PLoS ONE ◽

10.1371/journal.pone.0261215 ◽

2021 ◽

Vol 16 (12) ◽

pp. e0261215

Author(s):

Qurat-ul ain-Ali ◽

Nida Mushtaq ◽

Rabia Amir ◽

Alvina Gul ◽

Muhammad Tahir ◽

...

Keyword(s):

Abiotic Stress ◽

Gene Family ◽

Stress Responses ◽

Promoter Analysis ◽

Molecular Mechanisms ◽

Regulatory Elements ◽

Start Codon ◽

Gene Promoters ◽

Responsive Element ◽

Dreb Gene

Dehydration Responsive Element Binding (DREB) regulates the expression of numerous stress-responsive genes, and hence plays a pivotal role in abiotic stress responses and tolerance in plants. The study aimed to develop a complete overview of the cis-acting regulatory elements (CAREs) present in S. tuberosum DREB gene promoters. A total of one hundred and four (104) cis-regulatory elements (CREs) were identified from 2.5kbp upstream of the start codon (ATG). The in-silico promoter analysis revealed variable sets of cis-elements and functional diversity with the predominance of light-responsive (30%), development-related (20%), abiotic stress-responsive (14%), and hormone-responsive (12%) elements in StDREBs. Among them, two light-responsive elements (Box-4 and G-box) were predicted in 64 and 61 StDREB genes, respectively. Two development-related motifs (AAGAA-motif and as-1) were abundant in StDREB gene promoters. Most of the DREB genes contained one or more Myeloblastosis (MYB) and Myelocytometosis (MYC) elements associated with abiotic stress responses. Hormone-responsive element i.e. ABRE was found in 59 out of 66 StDREB genes, which implied their role in dehydration and salinity stress. Moreover, six proteins were chosen corresponding to A1-A6 StDREB subgroups for secondary structure analysis and three-dimensional protein modeling followed by model validation through PROCHECK server by Ramachandran Plot. The predicted models demonstrated >90% of the residues in the favorable region, which further ensured their reliability. The present study also anticipated pocket binding sites and disordered regions (DRs) to gain insights into the structural flexibility and functional annotation of StDREB proteins. The protein association network determined the interaction of six selected StDREB proteins with potato proteins encoded by other gene families such as MYB and NAC, suggesting their similar functional roles in biological and molecular pathways. Overall, our results provide fundamental information for future functional analysis to understand the precise molecular mechanisms of the DREB gene family in S. tuberosum.

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The DUB family in Populus: identification, characterization, evolution and expression patterns

BMC Genomics ◽

10.1186/s12864-021-07844-3 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Wenqing Zheng ◽

Liang Du

Keyword(s):

Abiotic Stress ◽

Stress Response ◽

Gene Structure ◽

Expression Profiles ◽

Populus Trichocarpa ◽

Expression Patterns ◽

Foliar Spray ◽

Regulatory Elements ◽

Limited Information ◽

Abiotic Stress Response

Abstract Background The deubiquitinase (DUB) family constitutes a group of proteases that regulate the stability or reverse the ubiquitination of many proteins in the cell. These enzymes participate in cell-cycle regulation, cell division and differentiation, diverse physiological activities such as DNA damage repair, growth and development, and response to stress. However, limited information is available on this family of genes in woody plants. Results In the present study, 88 DUB family genes were identified in the woody model plant Populus trichocarpa, comprising 44 PtrUBP, 3 PtrUCH, 23 PtrOTU, 4 PtrMJD, and 14 PtrJAMM genes with similar domains. According to phylogenetic analysis, the PtrUBP genes were classified into 16 groups, the PtrUCH genes into two, the PtrOTU genes into eight, the PtrMJD genes into two, and the PtrJAMM genes into seven. Members of same subfamily had similar gene structure and motif distribution characteristics. Synteny analysis of the DUB family genes from P. thrchocarpa and four other plant species provided insight into the evolutionary traits of DUB genes. Expression profiles derived from previously published transcriptome data revealed distinct expression patterns of DUB genes in various tissues. On the basis of the results of analysis of promoter cis-regulatory elements, we selected 16 representative PtrUBP genes to treatment with abscisic acid, methyl jasmonate, or salicylic acid applied as a foliar spray. The majority of PtrUBP genes were upregulated in response to the phytohormone treatments, which implied that the genes play potential roles in abiotic stress response in Populus. Conclusions The results of this study broaden our understanding of the DUB family in plants. Analysis of the gene structure, conserved elements, and expression patterns of the DUB family provides a solid foundation for exploration of their specific functions in Populus and to elucidate the potential role of PtrUBP gene in abiotic stress response.

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In silico promoter and expression analyses of rice Auxin Binding Protein 57 (ABP57)

Plant Science Today ◽

10.14719/pst.2021.8.3.1208 ◽

2021 ◽

Vol 8 (3) ◽

pp. 741-748

Author(s):

Farah Afiqah Baharuddin ◽

Zhan Xuan Khong ◽

Zamri Zainal ◽

Noor Liyana Sukiran

Keyword(s):

Abiotic Stress ◽

In Silico ◽

Binding Protein ◽

Transcript Level ◽

Regulatory Elements ◽

Biochemical Properties ◽

Auxin Receptor ◽

Reverse Transcription Pcr ◽

Auxin Binding ◽

Auxin Binding Protein

Auxin Binding Protein 57 (ABP57) is one of the molecular components involved in rice response to abiotic stress. The ABP57 gene encodes an auxin receptor which functions in activating the plasma membrane H+-ATPase. Biochemical properties of ABP57 have been characterized; however, the function of ABP57, particularly on stress and hormone responses is still limited. This study was conducted to understand the regulation of ABP57 expression under abiotic stress. Thus, in silico identification of cis-acting regulatory elements (CAREs) in the promoter region of ABP57 was performed. Several motifs and transcription factor binding site (TFBS) that are involved in abiotic stress such as ABRE, DRE, AP2/EREBP, WRKY and NAC were identified. Next, expression analysis of ABP57 under drought, salt, auxin (IAA) and abscisic acid (ABA) was conducted by reverse transcription-PCR (RT-PCR) to verify the effect of these treatments on ABP57 transcript level. ABP57 was expressed at different levels in the shoot and root under drought conditions, and its expression was increased under IAA and ABA treatments. Moreover, our results showed that ABP57 expression in the root was more responsive to drought, auxin and ABA treatments compared to its transcript in the shoot. This finding suggests that ABP57 is a drought-responsive gene and possibly regulated by IAA and ABA.

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RETRACTED ARTICLE: The specific MYB binding sites bound by TaMYB in the GAPCp2/3 promoters are involved in the drought stress response in wheat

BMC Plant Biology ◽

10.1186/s12870-019-1948-y ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 5

Author(s):

Lin Zhang ◽

Zhiqiang Song ◽

Fangfang Li ◽

Xixi Li ◽

Haikun Ji ◽

...

Keyword(s):

Gene Expression ◽

Abiotic Stress ◽

Drought Stress ◽

Reporter Gene ◽

Binding Sites ◽

Promoter Sequence ◽

Regulatory Elements ◽

Cis Elements ◽

Gus Reporter ◽

Almost All

Abstract Background Drought stress is one of the major abiotic stresses that affects plant growth and productivity. The GAPCp genes play important roles in drought stress tolerance in multiple species. The aim of this experiment was to identify the core cis-regulatory elements that may respond to drought stress in the GAPCp2 and GAPCp3 promoter sequences. Results In this study, the promoters of GAPCp2 and GAPCp3 were cloned. The promoter activities were significantly improved under abiotic stress via regulation of Rluc reporter gene expression, while promoter sequence analysis indicated that these fragments were not almost identical. In transgenic Arabidopsis with the expression of the GUS reporter gene under the control of one of these promoters, the activities of GUS were strong in almost all tissues except the seeds, and the activities were induced after abiotic stress. The yeast one-hybrid system and EMSA demonstrated that TaMYB bound TaGAPCp2P/3P. By analyzing different 5′ deletion mutants of these promoters, it was determined that TaGAPCp2P (− 1312~ − 528) and TaGAPCp3P (− 2049~ − 610), including the MYB binding site, contained enhancer elements that increased gene expression levels under drought stress. We used an effector and a reporter to co-transform tobacco and found that TaMYB interacted with the specific MYB binding sites of TaGAPCp2P (− 1197~ − 635) and TaGAPCp3P (− 1456~ − 1144 and − 718~ − 610) in plant cells. Then, the Y1H system and EMSA assay demonstrated that these MYB binding sites in TaGAPCp2P (− 1135 and − 985) and TaGAPCp3P (− 1414 and − 665) were the target cis-elements of TaMYB. The deletion of the specific MYB binding sites in the promoter fragments significantly restrained the drought response, and these results confirmed that these MYB binding sites (AACTAAA/C) play vital roles in improving the transcription levels under drought stress. The results of qRT-PCR in wheat protoplasts transiently overexpressing TaMYB indicated that the expression of TaGAPCp2/3 induced by abiotic stress was upregulated by TaMYB. Conclusion The MYB binding sites (AACTAAA/C) in TaGAPCp2P/3P were identified as the key cis-elements for responding to drought stress and were bound by the transcription factor TaMYB.

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Rational Design and Testing of Anti-Knock Additives

Energies ◽

10.3390/en13184923 ◽

2020 ◽

Vol 13 (18) ◽

pp. 4923

Author(s):

Andrew D. Ure ◽

Manik K. Ghosh ◽

Maria Rappo ◽

Roland Dauphin ◽

Stephen Dooley

Keyword(s):

Rational Design ◽

Reaction Pathway ◽

Cetane Number ◽

Research Octane Number ◽

Ignition Quality ◽

Ignition Quality Tester ◽

Generic Design ◽

The Relationship ◽

Design And Testing

An innovative and informed methodology for the rational design and testing of anti-knock additives is reported. Interaction of the additives with OH● and HO2● is identified as the key reaction pathway by which non-metallic anti-knock additives are proposed to operate. Based on this mechanism, a set of generic design criteria for anti-knock additives is outlined. It is suggested that these additives should contain a weak X-H bond and form stable radical species after hydrogen atom abstraction. A set of molecular structural, thermodynamic, and kinetic quantities that pertain to the propensity of the additive to inhibit knock by this mechanism are identified and determined for a set of 12 phenolic model compounds. The series of structural analogues was carefully selected such that the physical thermodynamic and kinetic quantities could be systematically varied. The efficacy of these molecules as anti-knock additives was demonstrated through the determination of the research octane number (RON) and the derived cetane number(DCN), measured using an ignition quality tester (IQT), of a RON 95 gasoline treated with 1 mole % of the additive. The use of the IQT allows the anti-knock properties of potential additives to be studied on one tenth of the scale, compared to the analogous RON measurement. Using multiple linear regression, the relationship between DCN/RON and the theoretically determined quantities is studied. The overall methodology reported is proposed as an informed alternative to the non-directed experimental screening approach typically adopted in the development of fuel additives.

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Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1511027112 ◽

2015 ◽

Vol 112 (39) ◽

pp. 12181-12186 ◽

Cited By ~ 82

Author(s):

Chaoxian Bai ◽

Yang Zhang ◽

Xuejin Zhao ◽

Yiling Hu ◽

Sihai Xiang ◽

...

Keyword(s):

Quantitative Method ◽

Fluorescent Protein ◽

Single Cells ◽

Gene Clusters ◽

Regulatory Elements ◽

Streptomyces Avermitilis ◽

Systems Biotechnology ◽

Synthetic Promoters ◽

Expression Of Genes ◽

Green Fluorescent

There is a great demand for precisely quantitating the expression of genes of interest in synthetic and systems biotechnology as new and fascinating insights into the genetics of streptomycetes have come to light. Here, we developed, for the first time to our knowledge, a quantitative method based on flow cytometry and a superfolder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. Single cells of filamentous bacteria were obtained by releasing the protoplasts from the mycelium, and the dead cells could be distinguished from the viable ones by propidium iodide (PI) staining. With this sophisticated quantitative method, some 200 native or synthetic promoters and 200 ribosomal binding sites (RBSs) were characterized in a high-throughput format. Furthermore, an insulator (RiboJ) was recruited to eliminate the interference between promoters and RBSs and improve the modularity of regulatory elements. Seven synthetic promoters with gradient strength were successfully applied in a proof-of-principle approach to activate and overproduce the cryptic lycopene in a predictable manner in Streptomyces avermitilis. Our work therefore presents a quantitative strategy and universal synthetic modular regulatory elements, which will facilitate the functional optimization of gene clusters and the drug discovery process in Streptomyces.

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