Gateway Binary Vectors with the Bialaphos Resistance Gene,bar, as a Selection Marker for Plant Transformation

Background: Gene manipulation strategies including gene knockout and editing are becoming more sophisticated in terms of mechanism of action, efficacy and ease of use. In classical molecular methods of gene knockout, homologous arms are designed for induction of crossing over event in double strand DNA. Recently, CRISPR/Cas9 system has been emerged as a precise and powerful tool for gene targeting. In this effort, we aimed to generate a CRISPR/Cas9-based vector specific for targeting genes in Leishmania parasites. Methods: U6 and DHFR promoters and neomycin-resistance gene were amplified from genome of L. major (MHRO/IR/75/ER) and pEGFP-N1, respectively. U6 promoter was cloned in pX330 vector which is named as pX330-U6. DHFR promoter and neo resistance gene sequence fragments were fused using a combination of SOE (Splicing by overlap extension)-PCR and T/A cloning techniques. To generate pX-leish, fused fragments su-bcloned into the pX330-U6. Two sgRNAs were designed to target the gp63 gene and cloned in pX-leish. Results: The pX-leish vector was designed for simultaneous expression of cas9 and G418 resistance proteins along with a self-cleaving 2A peptide for efficient separation of the two proteins. In this study pX-leish was designed with 3 features: 1) Compatible promoters with Leishmania parasites. 2) Insertion of antibiotic selection marker 3) Designing an all-in-one vector containing all components required for CRISPR/Cas9 system. Conclusion: This modified system would be valuable in genome manipulation studies in Leishmania for vaccine research in future.

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New Selection Marker for Plant Transformation

Recombinant Gene Expression ◽

10.1385/1-59259-774-2:385 ◽

2004 ◽

pp. 385-396

Author(s):

Barbara Leyman ◽

Nelson Avonce ◽

Matthew Ramon ◽

Patrick Van Dijck ◽

Johan M. Thevelein ◽

...

Keyword(s):

Plant Transformation ◽

Selection Marker

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A reporter for noninvasively monitoring gene expression and plant transformation

Horticulture Research ◽

10.1038/s41438-020-00390-1 ◽

2020 ◽

Vol 7 (1) ◽

Cited By ~ 1

Author(s):

Yubing He ◽

Tao Zhang ◽

Hui Sun ◽

Huadong Zhan ◽

Yunde Zhao

Keyword(s):

Gene Expression ◽

Plant Transformation ◽

Protein Localization ◽

Fruit Tree ◽

Selection Marker ◽

Special Equipment ◽

Chemical Treatments ◽

Effective Selection ◽

Monitoring Gene Expression ◽

Large Crop

Abstract Reporters have been widely used to visualize gene expression, protein localization, and other cellular activities, but the commonly used reporters require special equipment, expensive chemicals, or invasive treatments. Here, we construct a new reporter RUBY that converts tyrosine to vividly red betalain, which is clearly visible to naked eyes without the need of using special equipment or chemical treatments. We show that RUBY can be used to noninvasively monitor gene expression in plants. Furthermore, we show that RUBY is an effective selection marker for transformation events in both rice and Arabidopsis. The new reporter will be especially useful for monitoring cellular activities in large crop plants such as a fruit tree under field conditions and for observing transformation and gene expression in tissue culture under sterile conditions.

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