scholarly journals Development of potent promoters that drive the efficient expression of genes in apple protoplasts

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xianpu Wang ◽  
Lili Xu ◽  
Xiuxia Liu ◽  
Li Xin ◽  
Shujing Wu ◽  
...  
Keyword(s):  
Transient Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Signaling Cascades ◽  
Biochemical Mechanism ◽  
Expression Of Genes ◽  
Efficient Expression ◽  
Biochemical Analyses ◽  
The Stability ◽  
Powerful Strategy

AbstractProtoplast transient expression is a powerful strategy for gene functional characterization, especially in biochemical mechanism studies. We herein developed a highly efficient transient expression system for apple protoplasts. The abilities of the Arabidopsis thaliana and Malus domestica ubiquitin-10 (AtUBQ10 and MdUBQ10) promoters to drive the expression of multiple genes were compared with that of the CaMV 35S promoter, and the results revealed that the AtUBQ10 and MdUBQ10 promoters were more efficient in apple protoplasts. With this system, we demonstrated that active AtMKK7ac could activate MAPK6/3/4 signaling cascades, which further regulated MdWRKY33 phosphorylation and stability in apple. Furthermore, the ligand-induced interaction between the immune receptor AtFLS2 and the coreceptor AtBAK1 was reconstituted in apple protoplasts. We also found that the stability of the bacterial effector AvrRpt2 was regulated by feedback involving auxin and the immune regulator RIN4. The system established herein will serve as a useful tool for the molecular and biochemical analyses of apple genes.

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

Functional characterization of a stilbene synthase gene using a transient expression system in planta

2008 ◽  
Vol 28 (4) ◽  
pp. 589-599 ◽  
Author(s):  
Jose Condori ◽  
Giuliana Medrano ◽  
Ganapathy Sivakumar ◽  
Vipin Nair ◽  
Carole Cramer ◽  
...  
Keyword(s):  
Transient Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Stilbene Synthase ◽  
In Planta ◽  
Transient Expression System ◽  
Stilbene Synthase Gene

scholarly journals A high-efficiency Agrobacterium-mediated transient expression system in the leaves of Artemisia annua L.

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yongpeng Li ◽  
Tiantian Chen ◽  
Wei Wang ◽  
Hang Liu ◽  
Xin Yan ◽  
...  
Keyword(s):  
Transient Expression ◽  
High Throughput ◽  
High Efficiency ◽  
Artemisia Annua ◽  
Expression System ◽  
Functional Characterization ◽  
Cauliflower Mosaic Virus ◽  
Transient Transformation ◽  
Transformation System ◽  
Transient Expression System

Abstract Background The Agrobacterium-mediated transient transformation, which proved effective in diverse plant species, has been widely applied for high-throughput gene function studies due to its simplicity, rapidity, and high efficiency. Despite the efforts have made on Artemisia annua transient expression, achieving high-throughput gene functional characterization basing on a fast and easy-manipulated transient transformation system in A. annua remains challenging. Results The first pair of true leaves of A. annua is an ideal candidate for Agrobacterium injection. EHA105 was the optimal strain that can be used for the development of the transient expression system. The supplementation of Triton X-100 at a concentration of 0.005% greatly improved the transient expression frequency. According to the histochemical β-Glucuronidase (GUS) staining assay, high transient expression level of the reporter gene (GUS) maintained at least a week. Dual-luciferase (Dual-LUC) transient assays showed that the activity of cauliflower mosaic virus 35S (CaMV35S) promoter and its derivates varied between A. annua and tobacco. In A. annua, the CaMV35S promoter had comparable activity with double CaMV35S promoter, while in tobacco, CaMV35S exhibited approximately 50% activity of double CaMV35S promoter. Otherwise, despite the CaMV35S promoter and double CaMV35S promoter from GoldenBraid Kit 2.0 displayed high activity strength in tobacco, they demonstrated a very low activity in transiently expressed A. annua. The activity of UBQ10 promoter and endogenous UBQb promoter was investigated as well. Additionally, using our transient expression system, the transactivation of AaGSW1 and AaORA on AaCYP71AV1 promoter was confirmed. Dual-LUC assays demonstrated that AaHD8 activated the expression of two glandular secreting trichomes-specific lipid transfer protein genes AaLTP1 and AaLTP2, indicating that AaLTP1 and AaLTP2 might serve as downstream components of AaHD8-involved glandular trichome initiation and cuticle formation, as well as artemisinin secretion in A. annua. Conclusions A simple, rapid, good-reproducibility, high-efficiency and low-cost transient transformation system in A. annua was developed. Our method offered a new way for gene functional characterization studies such as gene subcellular localization, promoter activity and transcription activation assays in A. annua, avoiding the aberrant phenotypes resulting from gene expression in a heterologous system.

scholarly journals Highly Efficient Protoplast Isolation and Transient Expression System for Functional Characterization of Flowering Related Genes in Cymbidium Orchids

2020 ◽  
Vol 21 (7) ◽  
pp. 2264 ◽  
Author(s):  
Rui Ren ◽  
Jie Gao ◽  
Chuqiao Lu ◽  
Yonglu Wei ◽  
Jianpeng Jin ◽  
...  
Keyword(s):  
Transient Expression ◽  
Transfection Efficiency ◽  
Expression System ◽  
Functional Characterization ◽  
Protoplast Isolation ◽  
Bimolecular Fluorescence Complementation ◽  
Dna Amount ◽  
Time Saving ◽  
Highly Efficient ◽  
Transient Expression System

Protoplast systems have been proven powerful tools in modern plant biology. However, successful preparation of abundant viable protoplasts remains a challenge for Cymbidium orchids. Herein, we established an efficient protoplast isolation protocol from orchid petals through optimization of enzymatic conditions. It requires optimal D-mannitol concentration (0.5 M), enzyme concentration (1.2 % (w/v) cellulose and 0.6 % (w/v) macerozyme) and digestion time (6 h). With this protocol, the highest yield (3.50 × 107/g fresh weight of orchid tissue) and viability (94.21%) of protoplasts were obtained from flower petals of Cymbidium. In addition, we achieved high transfection efficiency (80%) through the optimization of factors affecting polyethylene glycol (PEG)-mediated protoplast transfection including incubation time, final PEG4000 concentration and plasmid DNA amount. This highly efficient protoplast-based transient expression system (PTES) was further used for protein subcellular localization, bimolecular fluorescence complementation (BiFC) assay and gene regulation studies of flowering related genes in Cymbidium orchids. Taken together, our protoplast isolation and transfection protocol is highly efficient, stable and time-saving. It can be used for gene function and molecular analyses in orchids and other economically important monocot crops.

scholarly journals Transcriptional and translational S-box riboswitches differ in ligand-binding properties

2020 ◽  
Vol 295 (20) ◽  
pp. 6849-6860
Author(s):  
Divyaa Bhagdikar ◽  
Frank J. Grundy ◽  
Tina M. Henkin
Keyword(s):  
Ligand Binding ◽  
Mutational Analysis ◽  
Ligand Interaction ◽  
Internal Loop ◽  
Expression Of Genes ◽  
Loop Sequence ◽  
Biochemical Analyses ◽  
The Stability ◽  
Regulatory Decisions ◽  
Kinetics Of

There are a number of riboswitches that utilize the same ligand-binding domain to regulate transcription or translation. S-box (SAM-I) riboswitches, including the riboswitch present in the Bacillus subtilis metI gene, which encodes cystathionine γ-synthase, regulate the expression of genes involved in methionine metabolism in response to SAM, primarily at the level of transcriptional attenuation. A rarer class of S-box riboswitches is predicted to regulate translation initiation. Here we identified and characterized a translational S-box riboswitch in the metI gene from Desulfurispirillum indicum. The regulatory mechanisms of riboswitches are influenced by the kinetics of ligand interaction. The half-life of the translational D. indicum metI RNA–SAM complex is significantly shorter than that of the transcriptional B. subtilis metI RNA. This finding suggests that, unlike the transcriptional RNA, the translational metI riboswitch can make multiple reversible regulatory decisions. Comparison of both RNAs revealed that the second internal loop of helix P3 in the transcriptional RNA usually contains an A residue, whereas the translational RNA contains a C residue that is conserved in other S-box RNAs that are predicted to regulate translation. Mutational analysis indicated that the presence of an A or C residue correlates with RNA–SAM complex stability. Biochemical analyses indicate that the internal loop sequence critically determines the stability of the RNA–SAM complex by influencing the flexibility of residues involved in SAM binding and thereby affects the molecular mechanism of riboswitch function.

Functional characterization and structural modelling of peptidoglycan degrading β-N-acetyl-glucosaminidase from a dental isolate of Serratia marcescens

2020 ◽  
Vol 23 ◽  
Author(s):  
Aditi Rathee ◽  
Anil Panwar ◽  
Seema Kumari ◽  
Sanjay Chhibber ◽  
Ashok Kumar
Keyword(s):  
Functional Characterization ◽  
Major Change ◽  
Crystal Form ◽  
Bound State ◽  
Dynamics Simulation ◽  
Gram Positive ◽  
Structural Cell ◽  
Stability Structure ◽  
Arginine Residues ◽  
The Stability

Introduction:: Enzymatic degradation of peptidoglycan, a structural cell wall component of Gram-positive bacteria, has attracted considerable attention being a specific target for many known antibiotics. Methods:: Peptidoglycan hydrolases are involved in bacterial lysis through peptidoglycan degradation. β-N-acetylglucosaminidase, a peptidoglycan hydrolase, acts on O-glycosidic bonds formed by N-acetylglucosamine and N-acetyl muramic acid residues of peptidoglycan. Aim of present study was to study the action of β-N-acetylglucosaminidase, on methicillin- resistant Staphylococcus aureus (MRSA) and other Gram-negative bacteria. Results:: We investigated its dynamic behaviour using molecular dynamics simulation and observed that serine and alanine residues are involved in catalytic reaction in addition to aspartic acid, histidine, lysine and arginine residues. When simulated in its bound state, the RMSD values were found lesser than crystal form in the time stamp of 1000 picoseconds revealing its stability. Structure remained stably folded over 1000 picoseconds without undergoing any major change further confirming the stability of complex. Conclusion:: It can be concluded that enzymes belonging to this category can serve as a tool in eradicating Gram-positive pathogens and associated infections.

scholarly journals Prediction of Functional Consequences of Missense Mutations in ANO4 Gene

2021 ◽  
Vol 22 (5) ◽  
pp. 2732
Author(s):  
Nadine Reichhart ◽  
Vladimir M. Milenkovic ◽  
Christian H. Wetzel ◽  
Olaf Strauß
Keyword(s):  
Transmembrane Protein ◽  
Functional Characterization ◽  
Missense Mutations ◽  
Mutant Proteins ◽  
Functional Consequences ◽  
New Perspective ◽  
The Stability ◽  
Dynamics Simulations ◽  
And Function

The anoctamin (TMEM16) family of transmembrane protein consists of ten members in vertebrates, which act as Ca2+-dependent ion channels and/or Ca2+-dependent scramblases. ANO4 which is primarily expressed in the CNS and certain endocrine glands, has been associated with various neuronal disorders. Therefore, we focused our study on prioritizing missense mutations that are assumed to alter the structure and stability of ANO4 protein. We employed a wide array of evolution and structure based in silico prediction methods to identify potentially deleterious missense mutations in the ANO4 gene. Identified pathogenic mutations were then mapped to the modeled human ANO4 structure and the effects of missense mutations were studied on the atomic level using molecular dynamics simulations. Our data show that the G80A and A500T mutations significantly alter the stability of the mutant proteins, thus providing new perspective on the role of missense mutations in ANO4 gene. Results obtained in this study may help to identify disease associated mutations which affect ANO4 protein structure and function and might facilitate future functional characterization of ANO4.

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