An <i>in vitro</i> recombination method to convert restriction‐ and ligation‐independent expression vectors

Guo, Feng; Chiang, Ming-Yi; Wang, Yingtong; Zhang, Yuzhu

doi:10.1002/biot.200700170

Cited by 31 publications

(21 citation statements)

References 18 publications

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“…Coding sequences of AtWRI1 without start codon was cloned into pEZY19 (Guo et al, 2008) [pEZY19 was a gift from Yu-Zhu Zhang (Addgene plasmid # 18668)] using the Gateway ® cloning system in accordance with the manufacturer’s instructions. The decahistidine tagged AtWRI1 (His-AtWRI1) was transformed into chemically competent Rosetta TM (Merck, Darmstadt, Germany) host strain in accordance with the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

WRINKLED1 Is Subject to Evolutionary Conserved Negative Autoregulation

et al. 2019

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High accumulation of storage compounds such as oil and starch are economically important traits of most agricultural crops. The genetic network determining storage compounds composition in crops has been the target of many biotechnological endeavors. Especially WRINKLED1 (WRI1), a well-known key transcription factor involved in the allocation of carbon into oil, has attracted much interest. Here we investigate the presence of an autoregulatory system involving WRI1 through transient expression in Nicotiana benthamiana leaves. Different lengths of the Arabidopsis WRI1 promotor region were coupled to a GUS reporter gene and the activity was measured when combined with constitutive expression of different WRI1 homologs from Arabidopsis thaliana , oat ( Avena sativa L.), yellow nutsedge (Cyperus esculentus L.), and potato ( Solanum tuberosum L.). We could show that increasing levels of each WRI1 homolog reduced the transcriptional activity of the Arabidopsis WRI1 upstream region. Through structural analysis and domain swapping between oat and Arabidopsis WRI1, we were able to determine that the negative autoregulation was clearly dependent on the DNA-binding AP2-domains. A DNA/protein interaction assay showed that AtWRI1 is unable to bind to its corresponding upstream region indicating non-direct interaction in vivo . Taken together, our results demonstrate a negative feedback loop of WRI1 expression and that it is an indirect interaction most likely caused by downstream targets of WRI1. We also show that it is possible to release WRI1 expression from this autoregulation by creating semi-synthetic WRI1 homologs increasing the potential use of WRI1 in biotechnological applications.

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Section: Methodsmentioning

confidence: 99%

WRINKLED1 Is Subject to Evolutionary Conserved Negative Autoregulation

et al. 2019

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“…Multiple approaches were employed to identify interaction between virus CLCuMB and host GhSnRK1 protein. Host domain information was deduced from NCBI conserved domain database (Marchler-Bauer et al, 2016), InterPro at EMBL-EBI (Guo et al, 2008), PROSITE (Sigrist et al, 2012), and ThreaDom (Xue et al, 2013). After domain localization, three-dimensional structure of GhSnRK1, its domains and CLCuMB-βC1 were also predicted using I-TASSER (Zhang, 2008).…”

Section: Methodsmentioning

confidence: 99%

In silico Prediction and Validations of Domains Involved in Gossypium hirsutum SnRK1 Protein Interaction With Cotton Leaf Curl Multan Betasatellite Encoded βC1

et al. 2019

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Cotton leaf curl disease (CLCuD) caused by viruses of genus Begomovirus is a major constraint to cotton ( Gossypium hirsutum ) production in many cotton-growing regions of the world. Symptoms of the disease are caused by Cotton leaf curl Multan betasatellite (CLCuMB) that encodes a pathogenicity determinant protein, βC1. Here, we report the identification of interacting regions in βC1 protein by using computational approaches including sequence recognition, and binding site and interface prediction methods. We show the domain-level interactions based on the structural analysis of G. hirsutum SnRK1 protein and its domains with CLCuMB-βC1. To verify and validate the in silico predictions, three different experimental approaches, yeast two hybrid, bimolecular fluorescence complementation and pull down assay were used. Our results showed that ubiquitin-associated domain (UBA) and autoinhibitory sequence (AIS) domains of G. hirsutum -encoded SnRK1 are involved in CLCuMB-βC1 interaction. This is the first comprehensive investigation that combined in silico interaction prediction followed by experimental validation of interaction between CLCuMB-βC1 and a host protein. We demonstrated that data from computational biology could provide binding site information between CLCuD-associated viruses/satellites and new hosts that lack known binding site information for protein–protein interaction studies. Implications of these findings are discussed.

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“…The plasmid of pDONR221- ScAPX6 was digested with Ava I and then gel-purified for LR reaction with prokaryotic expressive vector of pEZYHb according to the manufacturer's instructions of LR Clonase™ II Enzyme Mix (Invitrogen, USA). The recombinant plasmid of pEZYHb-ScAPX6 was transformed into the competent cells E. coli BL21 (DE3) and then induced by 1.0 mmol·L −1 isopropyl β–D-thiogalactoside (IPTG) at 28°C for 0, 2, 4, and 8 h (Guo et al, 2008 ). LB medium with E. coli BL21 (blank) and BL21+pEZYHb (control) were induced by 1.0 mmol·L −1 IPTG for 0 and 8 h, respectively.…”

Section: Methodsmentioning

confidence: 99%

A Novel L-ascorbate Peroxidase 6 Gene, ScAPX6, Plays an Important Role in the Regulation of Response to Biotic and Abiotic Stresses in Sugarcane

et al. 2018

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The L-ascorbate peroxidase 6 gene (APX6) is one of the most important genes for scavenging H2O2 and plays a vital role in plant resistance to environmental stresses. In this study, a novel ScAPX6 gene (GenBank Accession No. KT907352) was obtained from a sugarcane variety (ROC22). Bioinformatics analysis showed that ScAPX6 has a cDNA length of 1,086 bp and encoded 333 amino acid residues. Subcellular localization confirmed that ScAPX6 was located in the chloroplast. Enhanced growth of Escherichia coli BL21 cells that expressed ScAPX6 showed high tolerance under copper (Cu) stress. Real-time quantitative PCR analysis revealed that ScAPX6 was constitutively expressed wherein with the highest expression levels in sugarcane pith and leaf and the lowest in the root. ScAPX6 was down-regulated by salicylic acid (SA), hydrogen peroxide (H2O2), polyethylene glycol (PEG) and sodium chloride (NaCl) stimuli. Interestingly, it was significantly up-regulated under the stresses of abscisic acid (ABA) and methyl jasmonate (MeJA) wherein with the highest inducible expression levels at 6 h at 6.0- and 70.0-times higher, respectively than that of control. Overexpression of ScAPX6 in Nicotiana benthamiana leaves enhanced the resistance to the infection of tobacco pathogens Pseudomonas solanacearum and Fusarium solani var. coeruleum. These results implied that ScAPX6 might positively respond to ABA, MeJA, and Cu, but might negatively respond to the stresses of SA, H2O2, PEG, and NaCl. Keeping in view the current investigation, ScAPX6 could be associated with the hypersensitive response (HR) or immunity of sugarcane, which will provide a baseline for the function identification of sugarcane ScAPX6.

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An in vitro recombination method to convert restriction‐ and ligation‐independent expression vectors

Cited by 31 publications

References 18 publications

WRINKLED1 Is Subject to Evolutionary Conserved Negative Autoregulation

WRINKLED1 Is Subject to Evolutionary Conserved Negative Autoregulation

In silico Prediction and Validations of Domains Involved in Gossypium hirsutum SnRK1 Protein Interaction With Cotton Leaf Curl Multan Betasatellite Encoded βC1

A Novel L-ascorbate Peroxidase 6 Gene, ScAPX6, Plays an Important Role in the Regulation of Response to Biotic and Abiotic Stresses in Sugarcane

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