scholarly journals Manipulating ZmEXPA4 expression ameliorates the drought-induced prolonged anthesis and silking interval in maize

2021 ◽  
Author(s):  
Boxin Liu ◽  
Bin Zhang ◽  
Zhirui Yang ◽  
Yan Liu ◽  
Shiping Yang ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Underlying Mechanism ◽  
Inducible Promoter ◽  
Environmental Threat ◽  
Trait Improvement ◽  
Comprehensive Development ◽  
Synchronous Development ◽  
Monoecious Plant ◽  
Significant Yield ◽  
Maize Grain

Abstract Drought poses a major environmental threat to maize (Zea mays) production worldwide. Since maize is a monoecious plant, maize grain yield is dependent on the synchronous development of male and female inflorescences. When a drought episode occurs during flowering, however, an asynchronism occurs in the anthesis and silking interval (ASI) that results in significant yield losses. The underlying mechanism responsible for this asynchronism is still unclear. Here, we obtained a comprehensive development-drought transcriptome atlas of maize ears. Genes that function in cell expansion and growth were highly repressed by drought in 50 mm ears. Notably, an association study using a natural-variation population of maize revealed a significant relationship between the level of α-expansin4 (ZmEXPA4) expression and drought-induced increases in ASI. Furthermore, genetic manipulation of ZmEXPA4 expression using a drought-inducible promoter in developing maize ears reduced the ASI under drought conditions. These findings provide important insights into the molecular mechanism underlying the increase in ASI in maize ears subjected to drought and provide a promising strategy that can be used for trait improvement.

scholarly journals ROS and glutathionylation balance cytoskeletal dynamics in neutrophil extracellular trap formation

2017 ◽  
Vol 216 (12) ◽  
pp. 4073-4090 ◽  
Author(s):  
Darko Stojkov ◽  
Poorya Amini ◽  
Kevin Oberson ◽  
Christiane Sokollik ◽  
Andrea Duppenthaler ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Genetic Defect ◽  
Underlying Mechanism ◽  
Neutrophil Extracellular Trap ◽  
Pharmacological Agents ◽  
Cytoskeletal Network ◽  
Cytoskeletal Dynamics ◽  
Dna Release ◽  
The One ◽  
Syndrome Protein

The antimicrobial defense activity of neutrophils partly depends on their ability to form neutrophil extracellular traps (NETs), but the underlying mechanism controlling NET formation remains unclear. We demonstrate that inhibiting cytoskeletal dynamics with pharmacological agents or by genetic manipulation prevents the degranulation of neutrophils and mitochondrial DNA release required for NET formation. Wiskott-Aldrich syndrome protein–deficient neutrophils are unable to polymerize actin and exhibit a block in both degranulation and DNA release. Similarly, neutrophils with a genetic defect in NADPH oxidase fail to induce either actin and tubulin polymerization or NET formation on activation. Moreover, neutrophils deficient in glutaredoxin 1 (Grx1), an enzyme required for deglutathionylation of actin and tubulin, are unable to polymerize either cytoskeletal network and fail to degranulate or release DNA. Collectively, cytoskeletal dynamics are achieved as a balance between reactive oxygen species–regulated effects on polymerization and glutathionylation on the one hand and the Grx1-mediated deglutathionylation that is required for NET formation on the other.

scholarly journals Efficient Genome Editing in Bacillus licheniformis Mediated by a Conditional CRISPR/Cas9 System

2020 ◽  
Vol 8 (5) ◽  
pp. 754
Author(s):  
Youran Li ◽  
Hanrong Wang ◽  
Liang Zhang ◽  
Zhongyang Ding ◽  
Sha Xu ◽  
...  
Keyword(s):  
Success Rate ◽  
Genome Editing ◽  
Bacillus Licheniformis ◽  
Genetic Manipulation ◽  
Inducible Promoter ◽  
Bacterial Cells ◽  
Protospacer Adjacent Motif ◽  
Constitutive Overexpression ◽  
Short Palindromic Repeat ◽  
Overexpression System

Bacillus licheniformis is widely used to produce multiple enzymes and chemicals in industrial fermentation. It is also an organism that is hard to genetically manipulate, which is mainly attributed to its extremely low transformation efficiency. The lack of genetic modification technology severely limits its further application. In this study, an all-in-one conditional clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 plasmid was developed for B. licheniformis with the cas9 gene under the control of a xylose-inducible promoter. By means of this design, the expression of the cas9 gene could be repressed without xylose, which significantly improved the transformation ratio from less than 0.1 cfu/μg to 2.42 cfu/μg DNA. Compared with this conditional system, a constitutive overexpression system led to significant growth retardation in bacterial cells. Both the biomass and specific growth rate decreased greatly. After transformation, successful genome editing could be triggered by 0.5% xylose. When the α-amylase gene amyL was used as a genomic target, the efficiencies of its disruption using three different protospacer-adjacent motif (PAM) sequences were 64.3%, 70.9%, and 47.1%, respectively. Moreover, temperature plays a pivotal role in the function of the constructed CRISPR system. The maximum success rate reached 97% at 20 °C, while higher temperatures negatively impacted the function of the system. These results suggested that the design with a cas9 gene under the strict control of a xylose-inducible promoter significantly improved the success rate of genome editing in this host. This work contributes to the development of genetic manipulation and furthers the use of B. licheniformis as an efficient industrial workhorse.

Effect of Temperature on Enzymes in the Pathway of Starch Biosynthesis in Developing Wheat and Maize Grain

1994 ◽  
Vol 21 (6) ◽  
pp. 807 ◽  
Author(s):  
PL Keeling ◽  
R Banisadr ◽  
L Barone ◽  
BP Wasserman ◽  
GW Singletary
Keyword(s):  
Elevated Temperature ◽  
Thermal Inactivation ◽  
Starch Synthesis ◽  
Soluble Starch ◽  
Effect Of Temperature ◽  
Branching Enzyme ◽  
Temperature Optimum ◽  
Significant Yield ◽  
Maize Grain ◽  
Lower Temperature

Soluble starch synthase (SSS) is shown to be a major site of control of flux through the pathway of starch synthesis in developing wheat and maize grain. Temperatures above 25�C adversely affect flux, and therefore, limit yield. This process is linked to SSS which is heat sensitive. Two apparently different properties of SSS can be identified which differ in the period required before full activity is restored after heat treatment. First, enzyme rate is adversely affected by elevated temperature, an effect which is reversible on returning to a lower temperature. The effect on enzyme rate was quantified using enzyme Q10 which was found to begin to be sub-optimal above 20�C. Second, with a prolonged period of exposu;e to elevated temperature there is a loss of enzyme activity which is not freely reversible which we have termed thermal inactivation. Although this occurs at temperatures in excess of 20�C in wheat, higher temperatures of more than 30�C are needed in maize SSS. Elevated temperature did not affect the inherent stability or Q10 characteristics of other enzymes in the pathway of starch synthesis except for branching enzyme which we believe has minimal flux-control strength. SSS thermal inactivation may not be a major problem in field conditions for developing maize grain, because temperatures rarely are high enough. However, we suggest that the effect on enzyme Q10 is more physiologically relevant, since maize SSS is operating sub-optimally as temperatures exceed 20�C. Calculations of the reductions in maize US corn-belt yield showed that significant yield improvement might be obtained by a 5�C shift in the temperature optimum. Thus selections for a more temperature tolerant form of maize SSS were conducted using enzyme Q10 as a selection tool. Of several hundred maize specimens screened, two were found to be significantly different. However, attempts to use backcross breeding to transfer this trait from the tropical donor to another line have not yet succeeded. Transgenic approaches to altering relations of starch deposition are now underway.

scholarly journals Genome Editing in Model Strain Myxococcus xanthus DK1622 by a Site-Specific Cre/loxP Recombination System

Biomolecules ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 137 ◽  
Author(s):  
Ying-Jie Yang ◽  
Raghvendra Singh ◽  
Xin Lan ◽  
Cheng-Sheng Zhang ◽  
Yue-Zhong Li ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Genetic Manipulation ◽  
Myxococcus Xanthus ◽  
Gene Clusters ◽  
Inducible Promoter ◽  
Engineering System ◽  
Suicide Vector ◽  
Recombination System ◽  
Gene Polymerase Chain Reaction ◽  
Deletion Frequency

Myxococcus xanthus DK1622 is a rich source of novel secondary metabolites, and it is often used as an expression host of exogenous biosynthetic gene clusters. However, the frequency of obtaining large genome-deletion variants by using traditional strategies is low, and progenies generated by homologous recombination contain irregular deletions. The present study aims to develop an efficient genome-engineering system for this bacterium based on the Cre/loxP system. We first verified the functionality of the native cre system that was integrated into the chromosome with an inducible promoter PcuoA. Then we assayed the deletion frequency of 8-bp-spacer-sequence mutants in loxP by Cre recombinase which was expressed by suicide vector pBJ113 or self-replicative vector pZJY41. It was found that higher guanine content in a spacer sequence had higher deletion frequency, and the self-replicative vector was more suitable for the Cre/loxP system, probably due to the leaky expression of inducible promoter PcuoA. We also inspected the effects of different antibiotics and the native or synthetic cre gene. Polymerase chain reaction (PCR) and sequencing of new genome joints confirmed that the Cre/loxP system was able to delete a 466 kb fragment in M. xanthus. This Cre/loxP-mediated recombination could serve as an alternative genetic manipulation method.

scholarly journals Inducible Selectable Marker Genes to Improve Aspergillus fumigatus Genetic Manipulation

2021 ◽  
Vol 7 (7) ◽  
pp. 506
Author(s):  
Clara Baldin ◽  
Alexander Kühbacher ◽  
Petra Merschak ◽  
Luis Enrique Sastré-Velásquez ◽  
Beate Abt ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Selectable Marker ◽  
Genetic Manipulation ◽  
Inducible Promoter ◽  
Fungal Species ◽  
Marker Genes ◽  
Selectable Markers ◽  
Simple Modification ◽  
Selectable Marker Genes

The hygromycin B phosphotransferase gene from Escherichia coli and the pyrithiamine resistance gene from Aspergillus oryzae are two dominant selectable marker genes widely used to genetically manipulate several fungal species. Despite the recent development of CRISPR/Cas9 and marker-free systems, in vitro molecular tools to study Aspergillus fumigatus, which is a saprophytic fungus causing life-threatening diseases in immunocompromised hosts, still rely extensively on the use of dominant selectable markers. The limited number of drug selectable markers is already a critical aspect, but the possibility that their introduction into a microorganism could induce enhanced virulence or undesired effects on metabolic behavior constitutes another problem. In this context, here, we demonstrate that the use of ptrA in A. fumigatus leads to the secretion of a compound that allows the recovery of thiamine auxotrophy. In this study, we developed a simple modification of the two commonly used dominant markers in which the development of resistance can be controlled by the xylose-inducible promoter PxylP from Penicillium chrysogenum. This strategy provides an easy solution to avoid undesired side effects, since the marker expression can be readily silenced when not required.

scholarly journals Regulation of the one carbon folate cycle as a shared metabolic signature of longevity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Andrea Annibal ◽  
Rebecca George Tharyan ◽  
Maribel Fides Schonewolff ◽  
Hannah Tam ◽  
Christian Latza ◽  
...  
Keyword(s):  
High Resolution Mass Spectrometry ◽  
Genetic Manipulation ◽  
Underlying Mechanism ◽  
Comparative Approach ◽  
Causal Mechanism ◽  
C Elegans ◽  
Health And Disease ◽  
One Carbon Metabolism ◽  
The One ◽  
Folate Cycle

AbstractThe metabolome represents a complex network of biological events that reflects the physiologic state of the organism in health and disease. Additionally, specific metabolites and metabolic signaling pathways have been shown to modulate animal ageing, but whether there are convergent mechanisms uniting these processes remains elusive. Here, we used high resolution mass spectrometry to obtain the metabolomic profiles of canonical longevity pathways in C. elegans to identify metabolites regulating life span. By leveraging the metabolomic profiles across pathways, we found that one carbon metabolism and the folate cycle are pervasively regulated in common. We observed similar changes in long-lived mouse models of reduced insulin/IGF signaling. Genetic manipulation of pathway enzymes and supplementation with one carbon metabolites in C. elegans reveal that regulation of the folate cycle represents a shared causal mechanism of longevity and proteoprotection. Such interventions impact the methionine cycle, and reveal methionine restriction as an underlying mechanism. This comparative approach reveals key metabolic nodes to enhance healthy ageing.

Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

Face Inversion Effect Emerges Under Critical Configural Discrepancy

2010 ◽  
Vol 69 (3) ◽  
pp. 161-167 ◽  
Author(s):  
Jisien Yang ◽  
Adrian Schwaninger
Keyword(s):  
Face Recognition ◽  
Face Processing ◽  
Underlying Mechanism ◽  
Inversion Effect ◽  
Configural Processing ◽  
Major Contributor ◽  
Face Inversion Effect ◽  
Configural Information ◽  
Face Inversion ◽  
The Face

Configural processing has been considered the major contributor to the face inversion effect (FIE) in face recognition. However, most researchers have only obtained the FIE with one specific ratio of configural alteration. It remains unclear whether the ratio of configural alteration itself can mediate the occurrence of the FIE. We aimed to clarify this issue by manipulating the configural information parametrically using six different ratios, ranging from 4% to 24%. Participants were asked to judge whether a pair of faces were entirely identical or different. The paired faces that were to be compared were presented either simultaneously (Experiment 1) or sequentially (Experiment 2). Both experiments revealed that the FIE was observed only when the ratio of configural alteration was in the intermediate range. These results indicate that even though the FIE has been frequently adopted as an index to examine the underlying mechanism of face processing, the emergence of the FIE is not robust with any configural alteration but dependent on the ratio of configural alteration.

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