Optimization of transient gene expression in tomato (Solanum lycopersicum L.)

Document Type : Research Paper

Authors

1 PhD student of Plant Biotechnology, Department of Agronomy, Faculty of Agriculture, University of Kurdistan, Sanandaj, Iran

2 Associate Professor of Agricultural Biotechnology, Department of Plant production and Genetics, Faculty of Agriculture, University of Kurdistan, Sanandaj, Iran

3 Professor of Molecular Genetics and Genetics Engineering, Department of Agricultural Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran

4 Assistant Professor Dryland Agricultural Research Institute, Agricultural Research, Education and Extension Organization (ARREO), Maragheh, Iran

Abstract

Abstract
Objective
The aim of this study is to optimize tomato as a suitable plant system for transient gene transformation for the production of pharmaceutical and industrial compounds.
Materials and methods
In order to investigate transient gene expression, three concentrations of Agrobacterium in OD600 (0.4, 0.6 and 0.8), three times after inoculation (4, 7 and 10 days) and two conditions with the presence of p19 gene and without p19 were selected as factors affecting the gene expression. The vectors used in this research pXK2FS7 and pCAMBIA1304 contained GFP and P19 genes, respectively. For transient GFP transformation, leaves of seedlings were inoculated using agroinfiltration method. In this way, different concentrations of Agrobacterium containing pXK2FS7 vector and P19 vector were mixed together and the expression of GFP gene was investigated. All the leaves of tomato plants at the appropriate stage were injected by insulin syringe without needle on the lower surface of the leaf. In order to confirm the transfection of the GFP and its expression, RNA extraction, cDNA synthesis and the analysis of expression by Real-Time PCR were performed and the expression of the GFP protein with a fluorescence microscope was finally detected.
Results
The results showed that the concentration of Agrobacteria, the number of days after inoculation, and the expression of the p19 had significant effects on the GFP expression. The expression of p19 as a suppressor of gene silencing mechanism led to higher GFP expression in comparison with the absence of p19. Additionally, it was found that dilution of 0.6OD600 Agrobacterium was the most suitable concentration for high expression of GFP agroinfiltration. Furthermore, the highest level of GFP expression was obtained 7 days after leaf inoculation. The best combination for maximum GFP expression at transcript level obtained at 0.6 OD600, 7 days after inoculation and expression of p19.
Conclusions
The results of this research can be used in preliminary studies or optimization of recombinant protein production in tomato leaves.

Keywords

References

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Agricultural Biotechnology Journal
Volume 15, Issue 4
December 2023
Pages 1-22

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