Protocol: Streamline cloning of genes into binary vectors in Agrobacterium via the Gateway® TOPO vector system

<p>Abstract</p> <p>Background</p> <p>In plant functional genomic studies, gene cloning into binary vectors for plant transformation is a routine procedure. Traditionally, gene cloning has relied on restriction enzyme digestion and ligation. In recent years, however, Gateway^®cloning technology (Invitrogen Co.) has developed a fast and reliable alternative cloning methodology which uses a phage recombination strategy. While many Gateway- compatible vectors are available, we frequently encounter problems in which antibiotic resistance genes for bacterial selection are the same between recombinant vectors. Under these conditions, it is difficult, if not sometimes impossible, to use antibiotic resistance in selecting the desired transformants. We have, therefore, developed a practical procedure to solve this problem.</p> <p>Results</p> <p>An integrated protocol for cloning genes of interest from PCR to <it>Agrobacterium </it>transformants via the Gateway^®System was developed. The protocol takes advantage of unique characteristics of the replication origins of plasmids used and eliminates the necessity for restriction enzyme digestion in plasmid selections.</p> <p>Conclusion</p> <p>The protocol presented here is a streamlined procedure for fast and reliable cloning of genes of interest from PCR to <it>Agrobacterium </it>via the Gateway^®System. This protocol overcomes a key problem in which two recombinant vectors carry the same antibiotic selection marker. In addition, the protocol could be adapted for high-throughput applications.</p

Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling

BACKGROUND: The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A) tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A) tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. RESULTS: By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (approximately 17%) showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. CONCLUSION: An extensive protein network was revealed for plant polyadenylation machinery, in which all predicted proteins were found to be connecting to the complex. The gene expression profiles are indicative that specialized sub-complexes may be formed to carry out targeted processing of mRNA in different developmental stages and tissue types. These results offer a roadmap for further functional characterizations of the protein factors, and for building models when testing the genetic contributions of these genes in plant growth and development

A Polyadenylation Factor Subunit Implicated in Regulating Oxidative Signaling in Arabidopsis thaliana

BACKGROUND: Plants respond to many unfavorable environmental conditions via signaling mediated by altered levels of various reactive oxygen species (ROS). To gain additional insight into oxidative signaling responses, Arabidopsis mutants that exhibited tolerance to oxidative stress were isolated. We describe herein the isolation and characterization of one such mutant, oxt6. METHODOLOGY/PRINCIPAL FINDINGS: The oxt6 mutation is due to the disruption of a complex gene (At1g30460) that encodes the Arabidopsis ortholog of the 30-kD subunit of the cleavage and polyadenylation specificity factor (CPSF30) as well as a larger, related 65-kD protein. Expression of mRNAs encoding Arabidopsis CPSF30 alone was able to restore wild-type growth and stress susceptibility to the oxt6 mutant. Transcriptional profiling and single gene expression studies show elevated constitutive expression of a subset of genes that encode proteins containing thioredoxin- and glutaredoxin-related domains in the oxt6 mutant, suggesting that stress can be ameliorated by these gene classes. Bulk poly(A) tail length was not seemingly affected in the oxt6 mutant, but poly(A) site selection was different, indicating a subtle effect on polyadenylation in the mutant. CONCLUSIONS/SIGNIFICANCE: These results implicate the Arabidopsis CPSF30 protein in the posttranscriptional control of the responses of plants to stress, and in particular to the expression of a set of genes that suffices to confer tolerance to oxidative stress

Exploitation of dimension-dependent behavior of piezoelectric metamaterial with LC shunt circuit

To release the potential of piezoelectric metamaterial with LC local resonance, in this research we discuss its dimension-dependent behavior for the first time. It's found that the bandgap behavior is primarily related to the length/width of the piezoelectric transducers. In particular, the bandgap width of the piezoelectric metamaterial can be increased by 3.01 times by optimizing the transducers which has constant area of 64 mm2 and height of 0.5 mm. Moreover, it's illustrated that optimization of the dimension of transducer may yield better wave attenuation capability than adopting larger transducers. The analysis of the dimension-dependent behavior here is potential to be applied in the optimization of piezoelectric metamaterial

Arginine Decarboxylase Gene ADC2 Regulates Fiber Elongation in Cotton

Cotton is an important agro-industrial crop providing raw material for the textile industry. Fiber length is the key factor that directly affects fiber quality. ADC, arginine decarboxylase, is the key rate-limiting enzyme in the polyamine synthesis pathway; whereas, there is no experimental evidence that ADC is involved in fiber development in cotton yet. Our transcriptome analysis of the fiber initiation material of Gossypium arboreum L. showed that the expression profile of GaADC2 was induced significantly. Here, GhADC2, the allele of GaADC2 in tetraploid upland cotton Gossypium hirsutum L., exhibited up-regulated expression pattern during fiber elongation in cotton. Levels of polyamine are correlated with fiber elongation; especially, the amount of putrescine regulated by ADC was increased. Scanning electron microscopy showed that the fiber length was increased with exogenous addition of an ADC substrate or product putrescine; whereas, the fiber density was decreased with exogenous addition of an ADC specific inhibitor. Next, genome-wide transcriptome profiling of fiber elongation with exogenous putrescine addition was performed to determine the molecular basis in Gossypium hirsutum. A total of 3163 differentially expressed genes were detected, which mainly participated in phenylpropanoid biosynthesis, fatty acid elongation, and sesquiterpenoid and triterpenoid biosynthesis pathways. Genes encoding transcription factors MYB109, WRKY1, and TCP14 were enriched. Therefore, these results suggested the ADC2 and putrescine involvement in the development and fiber elongation of G. hirsutum, and provides a basis for cotton fiber development research in future

A Modified DF2016 Criterion for the Fracture Modeling from Shear to Equibiaxial Tension

This study introduces a modified DF2016 criterion to model a ductile fracture of sheet metals from shear to equibiaxial tension. The DF2016 criterion is modified so that a material constant is equal to the fracture strain at equibiaxial tension, which can be easily measured by the bulging experiments. To evaluate the performance of the modified DF2016 criterion, experiments are conducted for QP980 with five different specimens with stress states from shear to equibiaxial tension. The plasticity of the steel is characterized by the Swift–Voce hardening law and the pDrucker function, which is calibrated with the inverse engineering approach. A fracture strain is measured by the XTOP digital image correlation system for all the specimens, including the bulging test. The modified DF2016 criterion is also calibrated with the inverse engineering approach. The predicted force–stroke curves are compared with experimental results to evaluate the performance of the modified DF2016 criterion on the fracture prediction from shear to equibiaxial tension. The comparison shows that the modified DF2016 criterion can model the onset of the ductile fracture with high accuracy in wide stress states from shear to plane strain tension. Moreover, the calibration of the modified DF2016 criterion is comparatively easier than the original DF2016 criterion

Broadening Bandgap Width of Piezoelectric Metamaterial by Introducing Cavity

In this research, a semi-analytical model of the adaptive piezoelectric metamaterial, built upon continuum mechanics characterization, was formulated and analyzed to reveal the fundamental features of bandgap with respect to unit-cell parameters under transverse wave. A new mechanism to broaden the bandgap width, was then introduced through geometric cavity synthesis. It was demonstrated that the cavities incorporated into the host structure of the piezoelectric metamaterial can increase the electro-mechanical coupling of the system, which effectively yields broadened bandgap width. Case studies were performed to demonstrate the enhanced performance of the new design, as well as the tunability. Compared with the conventional piezoelectric metamaterial, the metamaterial with cavity synthesis can increase the bandgap width from 45 Hz to 126.7 Hz

Effective Convolutional Transformer for Highly Accurate Planetary Gearbox Fault Diagnosis

To extract the global temporal correlations and local features together to enhance the accuracy for fault diagnosis, this paper proposes an effective convolutional Transformer (ECT), which can learn the global temporal correlations using Transformer and local features with convolution at the same time. The proposed method designs a multi-stage hierarchical structure of Transformer, which utilizes convolutional tokenization to distill dominating sequence features from raw vibration signals while increasing the dimension of token embedding across stages at the same time as that in CNNs. The spatial-reduction attention (SRA) and the linear dimension reduction projections are introduced respectively to Transformer at different stages to reduce the resource consumption of the model. Finally, the proposed method utilizes a sequence pooling strategy on the output of Transformer to eliminate the requirement of the class token and make the model accurate for classification. The specially designed structure makes the model flexible and effective for planetary gearbox fault diagnosis. Experiments performed on planetary gearbox fault simulators indicate that the ECT method has significant effectiveness and high accuracy compared with the state-of-the-art methods for planetary gearbox fault diagnosis

Synergistic Regulation of Al Alloy Anode/Electrolyte Interface Layer in Al-Air Battery by Composite Inhibitor HEC‑K₂SnO₃

The extreme corrosion reaction of the Al anode in alkaline electrolyte is the biggest challenge for the industrialization of Al-air batteries (AABs). This article proposes a composite corrosion inhibitor composed of hydroxyethyl fiber and potassium stannate to weaken the corrosion reaction of Al anodes. The results showed that the hydrogen evolution rate suddenly decreased from 0.47 to 0.08 mL·cm–2·min–1 with the addition of composite corrosion inhibitors. The AABs with composite inhibitors exhibit a high energy density of 3311.26 mWh·g–1 and a power density of 75.0 mW·cm–2. Introducing hydroxyethyl cellulose (HEC) into an electrolyte containing K2SnO3, its rich O heteroatoms will adjust the growth state of Sn through adsorption, resulting in uniform deposition on the Al alloy anode. In addition, the polar hydroxyl groups in HEC are prone to forming organic Al salts (RO-Al) with Al3+ in solution, greatly activating the Al alloy anode. In summary, this review elaborates on the corrosion inhibition mechanisms of HEC, K2SnO3, and composite corrosion inhibitors, opening up prospects for the subsequent development of simple and effective organic, inorganic corrosion inhibitors