scholarly journals Efficiency of Recombinant CRISPR/rCas9-Mediated miRNA Gene Editing in Rice

2020 ◽  
Vol 21 (24) ◽  
pp. 9606
Author(s):  
Pil Joong Chung ◽  
Hoyong Chung ◽  
Nuri Oh ◽  
Joohee Choi ◽  
Seung Woon Bang ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Mutation Frequency ◽  
Function Analysis ◽  
Mirna Gene ◽  
Crop Productivity ◽  
Drought Response ◽  
Loss Of Function ◽  
Rice Plants ◽  
Guide Rnas ◽  
Base Insertion

Drought is one of the major environmental stresses adversely affecting crop productivity worldwide. Precise characterization of genes involved in drought response is necessary to develop new crop varieties with enhanced drought tolerance. Previously, we identified 66 drought-induced miRNAs in rice plants. For the further functional investigation of the miRNAs, we applied recombinant codon-optimized Cas9 (rCas9) for rice with single-guide RNAs specifically targeting mature miRNA sequences or sites required for the biogenesis of mature miRNA. A total of 458 T0 transgenic plants were analyzed to determine the frequency and type of mutations induced by CRISPR/rCas9 on 13 independent target miRNAs. The average mutation frequency for 13 genes targeted by single guide RNAs (sgRNAs) in T0 generation was 59.4%, including mono-allelic (8.54%), bi-allelic (11.1%), and hetero-allelic combination (39.7%) mutations. The mutation frequency showed a positive correlation with Tm temperature of sgRNAs. For base insertion, one base insertion (99%) was predominantly detected in transgenic plants. Similarly, one base deletion accounted for the highest percentage, but there was also a significant percentage of cases in which more than one base was deleted. The deletion of more than two bases in OsmiR171f and OsmiR818b significantly reduced the level of corresponding mature miRNAs. Further functional analysis using CRISPR/Cas9-mediated mutagenesis confirmed that OsmiR818b is involved in drought response in rice plants. Overall, this study suggests that the CRISPR/rCas9 system is a powerful tool for loss-of-function analysis of miRNA in rice.

scholarly journals First genome edited poinsettias: targeted mutagenesis of flavonoid 3′-hydroxylase using CRISPR/Cas9 results in a colour shift

2021 ◽  
Author(s):  
Daria Nitarska ◽  
Robert Boehm ◽  
Thomas Debener ◽  
Rares Calin Lucaciu ◽  
Heidi Halbwirth
Keyword(s):  
Transgenic Plants ◽  
Genome Editing ◽  
Ornamental Plants ◽  
Targeted Mutagenesis ◽  
Cloning And Expression ◽  
Euphorbia Pulcherrima ◽  
Loss Of Function ◽  
Wild Type ◽  
Promising Tool ◽  
Reddish Orange

AbstractThe CRISPR/Cas9 system is a remarkably promising tool for targeted gene mutagenesis, and becoming ever more popular for modification of ornamental plants. In this study we performed the knockout of flavonoid 3′-hydroxylase (F3′H) with application of CRISPR/Cas9 in the red flowering poinsettia (Euphorbia pulcherrima) cultivar ‘Christmas Eve’, in order to obtain plants with orange bract colour, which accumulate prevalently pelargonidin. F3′H is an enzyme that is necessary for formation of cyanidin type anthocyanins, which are responsible for the red colour of poinsettia bracts. Even though F3′H was not completely inactivated, the bract colour of transgenic plants changed from vivid red (RHS 45B) to vivid reddish orange (RHS 33A), and cyanidin levels decreased significantly compared with the wild type. In the genetically modified plants, an increased ratio of pelargonidin to cyanidin was observed. By cloning and expression of mutated proteins, the lack of F3′H activity was confirmed. This confirms that a loss of function mutation in the poinsettia F3′H gene is sufficient for obtaining poinsettia with orange bract colour. This is the first report of successful use of CRISPR/Cas9 for genome editing in poinsettia.

scholarly journals HMGB1 Promotes Systemic Lupus Erythematosus by Enhancing Macrophage Inflammatory Response

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Mudan Lu ◽  
Shanshan Yu ◽  
Wei Xu ◽  
Bo Gao ◽  
Sidong Xiong
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Inflammatory Response ◽  
Proinflammatory Cytokines ◽  
Lupus Erythematosus ◽  
Function Analysis ◽  
Critical Role ◽  
Loss Of Function ◽  
Systemic Lupus ◽  
Glycation End Products ◽  
Hmgb1 Expression

Background/Purpose. HMGB1, which may act as a proinflammatory mediator, has been proposed to contribute to the pathogenesis of multiple chronic inflammatory and autoimmune diseases including systemic lupus erythematosus (SLE); however, the precise mechanism of HMGB1 in the pathogenic process of SLE remains obscure.Method. The expression of HMGB1 was measured by ELISA and western blot. The ELISA was also applied to detect proinflammatory cytokines levels. Furthermore, nephritic pathology was evaluated by H&E staining of renal tissues.Results. In this study, we found that HMGB1 levels were significantly increased and correlated with SLE disease activity in both clinical patients and murine model. Furthermore, gain- and loss-of-function analysis showed that HMGB1 exacerbated the severity of SLE. Of note, the HMGB1 levels were found to be associated with the levels of proinflammatory cytokines such as TNF-αand IL-6 in SLE patients. Further study demonstrated that increased HMGB1 expression deteriorated the severity of SLE via enhancing macrophage inflammatory response. Moreover, we found that receptor of advanced glycation end products played a critical role in HMGB1-mediated macrophage inflammatory response.Conclusion. These findings suggested that HMGB1 might be a risk factor for SLE, and manipulation of HMGB1 signaling might provide a therapeutic strategy for SLE.

Identification of regulatory genes to improve nitrogen use efficiency

2014 ◽  
Vol 94 (6) ◽  
pp. 1009-1012 ◽  
Author(s):  
David R. Guevara ◽  
Yong-Mei Bi ◽  
Steven J. Rothstein
Keyword(s):  
Amino Acids ◽  
Transgenic Plants ◽  
Nitrogen Use Efficiency ◽  
Complex Traits ◽  
Regulatory Genes ◽  
Yield Potential ◽  
Environmental Damage ◽  
Nitrogen Use ◽  
Rice Plants ◽  
Use Efficiency

Guevara, D. R., Bi, Y.-M. and Rothstein, S. J. 2014. Identification of regulatory genes to improve nitrogen use efficiency. Can. J. Plant Sci. 94: 1009–1012. Crop production on soils containing sub-optimal levels of nitrogen (N) severely compromises yield potential. The development of crop varieties displaying high N use efficiency (NUE) is necessary in order to optimize N fertilizer use, and reduce the environmental damage caused by the current excessive application of N in agricultural areas. Genome-wide microarray analysis of rice plants grown under N-limiting environments was performed to identify NUE candidate genes. An early nodulin gene, OsENOD93-1, was strongly up-regulated during plant growth under low N. A constitutive Ubiquitin promoter was used to drive the expression of the OsENOD93-1 gene in transgenic plants to determine the importance of OsENOD93-1 for rice NUE. Transgenic rice plants over-expressing the OsENOD93-1 gene achieved ∼23% and 16% more yield and biomass, respectively, compared with wild-type plants when grown under N-limitation conditions. OsENOD93-1-OX transgenic plants accumulated a higher amount of total amino acids in the roots and xylem sap under N stress, suggesting that OsENOD93-1 plays a role in the transportation of amino acids. Taken together, we demonstrate that an effective way to identify NUE gene candidates involves both transcriptional profiling coupled with a transgenic validation approach to improve complex traits such as NUE in important crops.

scholarly journals When Coupled to Natural Transformation in Acinetobacter sp. Strain ADP1, PCR Mutagenesis Is Made Less Random by Mismatch Repair

2005 ◽  
Vol 71 (11) ◽  
pp. 7610-7612 ◽  
Author(s):  
Alison Buchan ◽  
L. Nicholas Ornston
Keyword(s):  
Structure Function ◽  
Mismatch Repair ◽  
Protein Function ◽  
Natural Transformation ◽  
Function Analysis ◽  
Repair System ◽  
Loss Of Function ◽  
Nucleotide Substitutions ◽  
Full Spectrum ◽  
Mismatch Repair System

ABSTRACT Random PCR mutagenesis is a powerful tool for structure-function analysis of targeted proteins, especially when coupled with DNA integration through natural transformation followed by selection for loss of function. The technique has been applied successfully to structure-function analysis of transcriptional regulators, enzymes, and transporters in Acinetobacter sp. strain ADP1. However, the mismatch repair system prevents the full spectrum of nucleotide substitutions that may be selected at the level of protein function from being recovered. This barrier may be overcome by introducing PCR-mutagenized genes into strains in which the corresponding genes have been deleted.

Jun mediates Frizzled-induced R3/R4 cell fate distinction and planar polarity determination in the Drosophila eye

Development ◽  
2000 ◽  
Vol 127 (16) ◽  
pp. 3619-3629 ◽  
Author(s):  
U. Weber ◽  
N. Paricio ◽  
M. Mlodzik
Keyword(s):  
Cell Fate ◽  
Genetic Interaction ◽  
Function Analysis ◽  
Loss Of Function ◽  
Planar Polarity ◽  
Jnk Signaling ◽  
Drosophila Eye ◽  
Polarity Determination ◽  
Signaling Components

Jun acts as a signal-regulated transcription factor in many cellular decisions, ranging from stress response to proliferation control and cell fate induction. Genetic interaction studies have suggested that Jun and JNK signaling are involved in Frizzled (Fz)-mediated planar polarity generation in the Drosophila eye. However, simple loss-of-function analysis of JNK signaling components did not show comparable planar polarity defects. To address the role of Jun and JNK in Fz signaling, we have used a combination of loss- and gain-of-function studies. Like Fz, Jun affects the bias between the R3/R4 photoreceptor pair that is critical for ommatidial polarity establishment. Detailed analysis of jun(−) clones reveals defects in R3 induction and planar polarity determination, whereas gain of Jun function induces the R3 fate and associated polarity phenotypes. We find also that affecting the levels of JNK signaling by either reduction or overexpression leads to planar polarity defects. Similarly, hypomorphic allelic combinations and overexpression of the negative JNK regulator Puckered causes planar polarity eye phenotypes, establishing that JNK acts in planar polarity signaling. The observation that Dl transcription in the early R3/R4 precursor cells is deregulated by Jun or Hep/JNKK activation, reminiscent of the effects seen with Fz overexpression, suggests that Jun is one of the transcription factors that mediates the effects of fz in planar polarity generation.

scholarly journals A systematic study of critical miRNAs on cells proliferation and apoptosis by the shortest path

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Peng Xu ◽  
Qian Wu ◽  
Deyang Lu ◽  
Jian Yu ◽  
Yongsheng Rao ◽  
...  
Keyword(s):  
Cell Fate ◽  
Shortest Path ◽  
Function Analysis ◽  
Mirna Gene ◽  
Small Noncoding Rnas ◽  
Liver Cancers ◽  
Proliferation And Apoptosis ◽  
Function Modules ◽  
Multiple Cell ◽  
The Impact

Abstract Background MicroRNAs are a class of important small noncoding RNAs, which have been reported to be involved in the processes of tumorigenesis and development by targeting a few genes. Existing studies show that the imbalance between cell proliferation and apoptosis is closely related to the initiation and development of cancers. However, the impact of miRNAs on this imbalance has not been studied systematically. Results In this study, we first construct a cell fate miRNA-gene regulatory network. Then, we propose a systematical method for calculating the global impact of miRNAs on cell fate genes based on the shortest path. Results on breast cancer and liver cancer datasets show that most of the cell fate genes are perturbed by the differentially expressed miRNAs. Most of the top-identified miRNAs are verified in the Human MicroRNA Disease Database (HMDD) and are related to breast and liver cancers. Function analysis shows that the top 20 miRNAs regulate multiple cell fate related function modules and interact tightly based on their functional similarity. Furthermore, more than half of them can promote sensitivity or induce resistance to some anti-cancer drugs. Besides, survival analysis demonstrates that the top-ranked miRNAs are significantly related to the overall survival time in the breast and liver cancers group. Conclusion In sum, this study can help to systematically study the important role of miRNAs on proliferation and apoptosis and thereby uncover the key miRNAs during the process of tumorigenesis. Furthermore, the results of this study will contribute to the development of clinical therapy based miRNAs for cancers.

scholarly journals Infection with an asymptomatic virus in rice results in a delayed drought response

2020 ◽  
Vol 47 (3) ◽  
pp. 239 ◽  
Author(s):  
Jaymee R. Encabo ◽  
Reena Jesusa A. Macalalad-Cabral ◽  
Jerlie Mhay K. Matres ◽  
Sapphire Charlene Thea P. Coronejo ◽  
Gilda B. Jonson ◽  
...  
Keyword(s):  
Drought Stress ◽  
Abiotic Stresses ◽  
Water Status ◽  
Drought Response ◽  
Plant Responses ◽  
Leaf Rolling ◽  
Rice Tungro Spherical Virus ◽  
Biotic And Abiotic Stresses ◽  
Rice Plants ◽  
Increased Susceptibility

Infection of viruses in plants often modifies plant responses to biotic and abiotic stresses. In the present study we examined the effects of Rice tungro spherical virus (RTSV) infection on drought response in rice. RTSV infection delayed the onset of leaf rolling by 1–2 days. During the delay in drought response, plants infected with RTSV showed higher stomatal conductance and less negative leaf water potential under drought than those of uninfected plants, indicating that RTSV-infected leaves were more hydrated. Other growth and physiological traits of plants under drought were not altered by infection with RTSV. An expression analysis of genes for drought response-related transcription factors showed that the expression of OsNAC6 and OsDREB2a was less activated by drought in RTSV-infected plants than in uninfected plants, further suggesting improved water status of the plants due to RTSV infection. RTSV accumulated more in plants under drought than in well-watered plants, indicating the increased susceptibility of rice plants to RTSV infection by drought. Collectively, these results indicated that infection with RTSV can transiently mitigate the influence of drought stress on rice plants by increasing leaf hydration, while drought increased the susceptibility of rice plants to RTSV.

Functional expression of heterologous proteins in yeast: insights into Ca2+ signaling and Ca2+-transporting ATPases

2004 ◽  
Vol 287 (3) ◽  
pp. C580-C589 ◽  
Author(s):  
Van-Khue Ton ◽  
Rajini Rao
Keyword(s):  
Secretory Pathway ◽  
Function Analysis ◽  
Model Organism ◽  
Functional Expression ◽  
Missense Mutations ◽  
Heterologous Proteins ◽  
Loss Of Function ◽  
Yeast Saccharomyces Cerevisiae ◽  
Culturing Conditions ◽  
Mutant Phenotypes

The baker's yeast Saccharomyces cerevisiae is a well-developed, versatile, and widely used model organism. It offers a compact and fully sequenced genome, tractable genetics, simple and inexpensive culturing conditions, and, importantly, a conservation of basic cellular machinery and signal transducing pathways with higher eukaryotes. In this review, we describe recent technical advances in the heterologous expression of proteins in yeast and illustrate their application to the study of the Ca2+ homeostasis machinery, with particular emphasis on Ca2+-transporting ATPases. Putative Ca2+-ATPases in the newly sequenced genomes of organisms such as parasites, plants, and vertebrates have been investigated by functional complementation of an engineered yeast strain lacking endogenous Ca2+ pumps. High-throughput screens of mutant phenotypes to identify side chains critical for ion transport and selectivity have facilitated structure-function analysis, and genomewide approaches may be used to dissect cellular pathways involved in Ca2+ transport and trafficking. The utility of the yeast system is demonstrated by rapid advances in the study of the emerging family of Golgi/secretory pathway Ca2+,Mn2+-ATPases (SPCA). Functional expression of human SPCA1 in yeast has provided insight into the physiology, novel biochemical characteristics, and subcellular localization of this pump. Haploinsufficiency of SPCA1 leads to Hailey-Hailey disease (HDD), a debilitating blistering disorder of the skin. Missense mutations, identified in patients with HHD, may be conveniently assessed in yeast for loss-of-function phenotypes associated with the disease.

scholarly journals Selective fertilization with phosphite allows unhindered growth of cotton plants expressing the ptxD gene while suppressing weeds

2018 ◽  
Vol 115 (29) ◽  
pp. E6946-E6955 ◽  
Author(s):  
Devendra Pandeya ◽  
Damar L. López-Arredondo ◽  
Madhusudhana R. Janga ◽  
LeAnne M. Campbell ◽  
Priscila Estrella-Hernández ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Sole Source ◽  
Crop Productivity ◽  
Weed Suppression ◽  
Artificial Substrates ◽  
Effective Alternative ◽  
Gene Gain ◽  
Weed Species ◽  
Weed Growth ◽  
Phosphite Dehydrogenase

Weeds, which have been the bane of agriculture since the beginning of civilization, are managed manually, mechanically, and, more recently, by chemicals. However, chemical control options are rapidly shrinking due to the recent rise in the number of herbicide-resistant weeds in crop fields, with few alternatives on the horizon. Therefore, there is an urgent need for alternative weed suppression systems to sustain crop productivity while reducing our dependence on herbicides and tillage. Such a development will also allay some of the negative perceptions associated with the use of herbicide-resistance genes and heavy dependence on herbicides. Transgenic plants expressing the bacterial phosphite dehydrogenase (ptxD) gene gain an ability to convert phosphite (Phi) into orthophosphate [Pi, the metabolizable form of phosphorus (P)]. Such plants allow for a selective fertilization scheme, based on Phi as the sole source of P for the crop, while offering an effective alternative for suppressing weed growth. Here, we show that, when P is supplied in the form of Phi, ptxD-expressing cotton (Gossypium hirsutum L.) plants outcompete, in both artificial substrates and natural soils from agricultural fields, three different monocot and dicot weed species intentionally introduced in the experiments, as well as weeds naturally present in the tested soils. Importantly, the ptxD/Phi system proved highly efficacious in inhibiting the growth of glyphosate-resistant Palmer amaranth. With over 250 weed species resistant to currently available herbicides, ptxD-transgenic plants fertilized with Phi could provide an effective alternative to suppressing the growth of these weeds while providing adequate nutrition to the crop.

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