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A New Method for Rapid Subcellular Localization and Gene Function Analysis in Cotton Based on Barley Stripe Mosaic
Weiwei Chen1,2, Chaolin Huang1,2, Chenmeng Luo1,2
1Zhengzhou Research Base, State Key Laboratory of Cotton Biology, School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, China.
Plants (Basel, Switzerland)
|July 9, 2022
Summary
A new Barley Stripe Mosaic Virus (BSMV) system efficiently overexpresses genes in cotton without genetic transformation. This virus-based method enables rapid gene function studies and DNA-free gene editing in cotton.
Area of Science:
- Plant molecular biology
- Genomics
- Biotechnology
Background:
- Genetic transformation in cotton is challenging, hindering functional genomics research.
- Virus-based vectors offer a promising alternative for rapid gene expression without transgene integration.
Purpose of the Study:
- To evaluate a novel four-component Barley Stripe Mosaic Virus (BSMV) system for gene overexpression in cotton.
- To assess the system's efficiency in infecting cotton and stably expressing large gene fragments.
Main Methods:
- Cotton seed imbibition with the BSMV vector.
- Systemic gene expression analysis using Green Fluorescent Protein (GFP) fusion.
- Subcellular localization studies using marker lines.
- Gene editing using split-SpCas9/sgRNA delivery.
- Overexpression of cotton Bile Acid Sodium Symporter (GhBASS5).
Main Results:
- The BSMV system successfully infected cotton via seed imbibition and achieved systemic overexpression of large gene fragments (up to 2340 bp).
- High-level expression of GFP-tagged genes was observed in various cotton tissues, with stable fluorescence for over 4 weeks.
- The system facilitated rapid establishment of subcellular localization marker lines and enabled co-delivery for protein interaction studies.
- GhBASS5 overexpression revealed its negative regulation of salt tolerance in cotton.
- DNA-free gene editing in cotton was achieved using the BSMV system.
Conclusions:
- The BSMV system is an efficient and rapid tool for gene overexpression in cotton, bypassing the need for genetic transformation.
- This virus-based approach significantly advances cotton functional genomics research and gene editing applications.
- The BSMV system supports diverse applications including subcellular localization, protein interaction studies, and gene editing.
