scholarly journals A Stable Genetic Transformation System and Implications of the Type IV Restriction System in the Nitrogen-Fixing Plant Endosymbiont Frankia alni ACN14a

2019 ◽  
Vol 10 ◽  
Author(s):  
Isaac Gifford ◽  
Summer Vance ◽  
Giang Nguyen ◽  
Alison M. Berry
Keyword(s):  
Genetic Transformation ◽  
Nitrogen Fixing ◽  
Transformation System ◽  
Restriction System ◽  
Type Iv ◽  
Frankia Alni ◽  
Genetic Transformation System

scholarly journals Stable Genetic Transformation and Heterologous Expression in the Nitrogen-fixing Plant Endosymbiont Frankia alni ACN14a

2018 ◽  
Author(s):  
Isaac Gifford ◽  
Summer Vance ◽  
Giang Nguyen ◽  
Alison M Berry
Keyword(s):  
Genetic Transformation ◽  
Root Nodule ◽  
Cell Types ◽  
Nitrogen Fixing ◽  
Chloramphenicol Resistance ◽  
Type Iv ◽  
Frankia Alni ◽  
Limited Culture ◽  
Nif Gene Cluster ◽  
Common Plant

Genus Frankia is comprised primarily of nitrogen-fixing actinobacteria that form root nodule symbioses with a group of hosts known as the actinorhizal plants. These plants are evolutionarily closely related to the legumes, which are nodulated by the rhizobia. Both host groups utilize homologs of nodulation genes for root-nodule symbiosis, derived from common plant ancestors. However the corresponding endosymbionts, Frankia and the rhizobia, are distantly related groups of bacteria, leading to questions of their symbiotic mechanisms and evolutionary history. To date, a stable system of genetic transformation has been lacking in Frankia. Here, we report the successful electrotransformation of Frankia alni ACN14a, by means of replicating plasmids expressing chloramphenicol-resistance for selection, and the use of GFP as a marker of gene expression. We have identified type IV methyl-directed restriction systems, highly-expressed in a range of actinobacteria, as a likely barrier to Frankia transformation and circumvented this barrier by using unmethylated plasmids, which allowed the transformation of F. alni as well as the maintenance of the plasmid. During nitrogen limitation, Frankia differentiates into two cell types: the vegetative hyphae and nitrogen-fixing vesicles. When the plasmid transformation system was used with expression of egfp under the control of the nif gene cluster promoter, it was possible to demonstrate by fluorescence imaging the expression of nitrogen fixation in vesicles but not hyphae in nitrogen-limited culture.

scholarly journals Establishment of a Genetic Transformation System in Guanophilic Fungus Amphichorda guana

2021 ◽  
Vol 7 (2) ◽  
pp. 138
Author(s):  
Min Liang ◽  
Wei Li ◽  
Landa Qi ◽  
Guocan Chen ◽  
Lei Cai ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Genetic Manipulation ◽  
Major Facilitator Superfamily ◽  
Protoplast Regeneration ◽  
Transformation System ◽  
Regeneration Rate ◽  
Bioactive Natural Products ◽  
Genetics Research ◽  
Genetic Transformation System ◽  
Major Facilitator

Fungi from unique environments exhibit special physiological characters and plenty of bioactive natural products. However, the recalcitrant genetics or poor transformation efficiencies prevent scientists from systematically studying molecular biological mechanisms and exploiting their metabolites. In this study, we targeted a guanophilic fungus Amphichorda guana LC5815 and developed a genetic transformation system. We firstly established an efficient protoplast preparing method by conditional optimization of sporulation and protoplast regeneration. The regeneration rate of the protoplast is up to about 34.6% with 0.8 M sucrose as the osmotic pressure stabilizer. To develop the genetic transformation, we used the polyethylene glycol-mediated protoplast transformation, and the testing gene AG04914 encoding a major facilitator superfamily transporter was deleted in strain LC5815, which proves the feasibility of this genetic manipulation system. Furthermore, a uridine/uracil auxotrophic strain was created by using a positive screening protocol with 5-fluoroorotic acid as a selective reagent. Finally, the genetic transformation system was successfully established in the guanophilic fungus strain LC5815, which lays the foundation for the molecular genetics research and will facilitate the exploitation of bioactive secondary metabolites in fungi.

Establishment of efficient callus genetic transformation system for Pyrus armeniacaefolia

2021 ◽  
Vol 289 ◽  
pp. 110429
Author(s):  
Xinhui Wang ◽  
Fengli Zhou ◽  
Jianlong Liu ◽  
Wenqian Liu ◽  
Shaoling Zhang ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Transformation System ◽  
Genetic Transformation System

Genetic transformation of Chainia and heat attenuation of its restriction system

1991 ◽  
Vol 37 (9) ◽  
pp. 713-715 ◽  
Author(s):  
Vijay M. Chauthaiwale ◽  
Pranav R. Vyas ◽  
Vasanti V. Deshpande
Keyword(s):  
Genetic Transformation ◽  
Key Words ◽  
Broad Host Range ◽  
Transformation System ◽  
Restriction System ◽  
Plasmid Replicon ◽  
Commercially Important ◽  
Low Efficiency ◽  
The Many ◽  
Restriction Modification

A PEG-mediated transformation system for Chainia (NCL 82-5-1) was develolped using a broad host range Streptomyces vector, pIJ702. Protoplasts prepared from Chainia (NCL 82-5-1) were regenerated with 5% efficiency. Transformation of the protoplasts with pIJ702 gave 10–20 transformants/μg DNA. The low efficiency of transformation is attributed to a restriction system in Chainia; this could be inhibited by treating the protoplasts at 42 °C for 10 min just before transformation. The yield of transformants increased 100-fold when pIJ702 was modified by passage in Chainia. Because the plasmid replicon was functional in Chainia and the modified plasmid was stably maintained, the transformation system should be useful for self-cloning in Chainia NCL 82-5-1 of the many commercially important enzymes this strain is known to produce. Key words: Chainia, transformation, Streptomyces, pIJ702 restriction modification, heat attenuation.

scholarly journals Establishment of a Tractable Genetic Transformation System in Veillonella spp.

2012 ◽  
Vol 78 (9) ◽  
pp. 3488-3491 ◽  
Author(s):  
Jinman Liu ◽  
Zhoujie Xie ◽  
Justin Merritt ◽  
Fengxia Qi
Keyword(s):  
Escherichia Coli ◽  
Genetic Transformation ◽  
Shuttle Vector ◽  
Transformation System ◽  
Genetic Studies ◽  
Content Type ◽  
Genetic Transformation System

ABSTRACTWe have constructed the firstEscherichia coli-Veillonellashuttle vector based on an endogenous plasmid (pVJL1) isolated from a clinicalVeillonellastrain. A highly transformableVeillonellastrain was also identified. Both the shuttle vector and the transformable strain should be valuable tools for futureVeillonellagenetic studies.

scholarly journals Robust Genetic Transformation System to Obtain Non-chimeric Transgenic Chickpea

2019 ◽  
Vol 10 ◽  
Author(s):  
Sudipta Shekhar Das Bhowmik ◽  
Alam Yen Cheng ◽  
Hao Long ◽  
Grace Zi Hao Tan ◽  
Thi My Linh Hoang ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Transformation System ◽  
Genetic Transformation System

scholarly journals Construction of a Genetic Transformation System for a Freeze-tolerant YeastKluyveromyces thermotolerans

1992 ◽  
Vol 56 (2) ◽  
pp. 228-232 ◽  
Author(s):  
Akihiro Hino ◽  
Chakamas Wongkhalaung ◽  
Shinya Kawai ◽  
Satoru Murao ◽  
Keiji Yano ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Transformation System ◽  
Freeze Tolerant ◽  
Genetic Transformation System
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