Project AP14869357: “The development of scientific basis for biotechnology for increasing the productivity of crops by modification of ribosomal protein S6 genes”.
Maim idea: We planed to clone cDNA-genes of two isoforms of ribosomal protein S6 from Arabidopsis thaliana (AtRPS6A and AtRPS6B) and replace the triplets encoding serines and threonine at the C-terminus of these proteins with a negatively charged glutamic acid codon using in vitro mutagenesis approach. Mutated cDNAs AtRPS6A(S231E,S237E,S240E,S241E,S247E,T249E) and AtRPS6B(S231E,S237E,S240E,S241E) will be integrated into the genome of potato plants as part of the DNA cassette of a binary vector. It is expected that the polypeptides encoded by these cDNA genes that mimic the phosphorylated form of AtRPS6 proteins, will activate the translation of the 5’TOP mRNA family and lead to an increase in ribosome biogenesis being integrated into the 40S ribosomal subunits. This should increase protein synthesis rates and enhance crop productivity under favorable conditions.
The goal is to develop the scientific basis for biotechnology for the production of genetically modified (GM) potato (Solanum tuberosum) plants with increased growth and maturation rates based on the expression of phosphomimetically mutated cDNA genes encoding ribosomal protein S6 from A. thaliana (AtRPS6A and AtRPS6B).
Project objectives:
1) To clone the cDNAs encoding two ribosomal protein S6 isoforms from A. thaliana (AtRPS6A and AtRPS6B) into the pET23c vector. To substitute the nucleotide triplets that encodes serine and threonine at the C-terminus of the AtRPS6A and AtRPS6B proteins to a glutamic acid codon.
2) To express mutated cDNAs in bacterial cells and detect synthetized recombinant proteins by immunoblotting. To purify recombinant His-AtRPS6A(S231E,S237E,S240E,S241E,S247E,T249E) and His-AtRPS6B(S231E,S237E,S240E,S241E) proteins by IMAC chromatography.
3) To insert variants of AtRPS6A and AtRPS6B cDNA genes with phosphomimetic substitutions into an agrobacterium binary vector.
4) To carry out transient expression of transgenes in plants for functional confirmation of their ability to be expressed in vivo.
5) To perform stable transformation of potato with constructs carrying mutated AtRPS6A or AtRPS6B cDNA variants. To check for the presence of transgenic inserts by PCR.
6) To evaluate the level of transgene expression in genetically modified potato plants. To compare biomass growth rates of control and GM potato plants expressing mutated variants of the AtRPS6A or AtRPS6B cDNA genes.
7) To evaluate the effect of the expression of AtRPS6A(S231E,S237E,S240E,S241E,S247E,T249E) and AtRPS6B(S231E,S237E,S240E,S241E) cDNA genes on polyribosome profiles in plant system in vivo.
The main results obtained were:
1) The AtRPS6A and AtRPS6B cDNA-genes from A. thaliana were cloned into the pET23c vector; in vitro mutagenesis of these cDNAs was performed to introduce phosphomimetic substitutions. The correctness of the introduced mutations was verified by sequencing.
2) The mutated cDNA genes were expressed in E. coli cells, the recombinant proteins were isolated by the IMAC method, and the integrity of the proteins was confirmed by immunoblotting.
3) Binary vectors 35P-AtRPS6A(S231E,S237E,S240E,S241E,S247E,T249E)-35T-pCAMBIA2300 and 35P-AtRPS6B(S231E,S237E,S240E,S241E)-35T-pCAMBIA2300 for transgene expression in plant cells were constructed.
4) Transient expression of transgenes was carried out in plants with confirmation of protein synthesis by immunoblotting.
5) Stable transformation of potatoes was carried out, regenerants with transgene inserts in the genome were obtained. During PCR analysis of regenerants, PCR product of the expected size was detected in five regenerants of the AtRPS6A(S231E,S237E,S240E,S241E,S247E,T249E) variant (12.5%; 5/40; 95% CI: 4.2-26.8%) and four regenerants of the AtRPS6B(S231E,S237E,S240E,S241E) variant (10.0%; 4/40; 95% CI: 2.8-23.7%) out of forty used explants.
6) Stable expression of transgenes in three potato regenerants was confirmed by RT-PCR, plant growth indicators were assessed. A positive response to the presence of the target RNA was detected only in two of five regenerants with the transgene insert 35P-AtRPS6A(S231E,S237E,S240E,S241E,S247E,T249E)-35T and only in one of four regenerants with the transgene insert 35P-AtRPS6B(S231E,S237E,S240E,S241E)-35T. The GM potato line with confirmed expression of the AtRPS6B(S231E,S237E,S240E,S241E) transgene S6-B-1 showed a significant increase in the number of leaves and total leaf surface area, but not in the length of stems and main roots, compared to the control plants.
7) A sedimentation analysis of ribosome-containing complexes in GM and control plants was carried out, and a comparison of ribosome profiles was made. The ratio of polysomes to non-polysomes in control plants (6.16 ± 0.09) did not differ significantly from plants expressing the AtRPS6A(S231E,S237E,S240E,S241E,S247E,T249E) transgene (6.70 ± 0.19; p = 0.0917) and from plants expressing the AtRPS6B(S231E,S237E,S240E,S241E) transgene (6.51 ± 0.32; p = 0.3189).
Publications and methodological developments
Publications for 2023:
1) Zhigailov A.V., Nizkorodova A.S., Sharipov K.O., Polimbetova, N. S., Iskakov B.K. Glyphosate treatment mediates the accumulation of small discrete 5′- and 3′-terminal fragments of 18S rRNA in plant cells. Vavilovskii zhurnal genetiki i selektsii. – 2023. – Vol. 27, No.2. – P. 93–98. https://doi.org/10.18699/VJGB-23-13. (Web of Science: Q3; Scopus: процентиль 51%, CiteScore 1.9).
2) Nadirova L.T., Beisenov D.K., Stanbekova G.E., A.V. Zhigaylov, B.K. Iskakov. Cloning and modification of cDNA genes of ribosomal protein S6 from Arabidopsis thaliana // Fundamental and applied research in the field of molecular biology, biochemistry, biotechnology. – Proceedings of the International Scientific Conference of Young Scientists dedicated to the 40th anniversary of the founding of the Institute of Molecular Biology and Biochemistry named after M.A. Aitkhozhin (November 19, 2023). – Almaty, 2023. – P. 32. (in Russian)
Publications for 2024:
3) Nadirova L.T., Beisenov D.K, Stanbekova G.E., Zhigailov A.V., Ryabova L.A., Iskakov B.K. Cloning of cDNA-gene of Arabidopsis thaliana ribosomal protein S6, its expression in Escherichia coli and purification of AtRPS6B1 recombinant protein // Eurasian Journal of Applied Biotechnology. – 2024. No.1. – P. 69-75. https://doi.org/10.11134/btp.1.2024.7. The journal is approved by the Science and Higher Education Quality Assurance Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan.
Laboratory regulations have been drawn up (2024):
“Method for obtaining genetically modified potato plants (Solanum tuberosum) expressing the AtRPS6B(S231E,S237E,S240E,S241E) transgene and their identification using the PCR method”.
Исполнители проекта (основной научный состав):
- Andrey Zhigailov, Associate professor, project supervisor, head of the Laboratory of Protein and Nucleic Acids. H-index: 5. ORCID ID: 0000-0002-9646-033X. Scopus ID: 6508121286. Web of science ID: N-6073-2015.
- Ruslan Kryldakov, Principal investigator, senior researcher. H-index: 3. ORCID ID: 0000-0002-2299-3109.
- Bulat Iskakov, Doctor of biology, professor, chief researcher. H-index: 6. ORCID ID: 0000-0002-5204-4377.
- Gulshan Stanbekova, Associate professor, leading researcher. H-index: 5. ORCID ID: 0000-0002-7819-6475.
- Oksana Karpova, Leading researcher. H-index: 3. ORCID ID: 0000-0001-9643-2913.
- Anna Nizkorodova, Associate professor, leading researcher. H-index: 2. ORCID ID: 0000-0002-1597-7207.
- Nailya Polimbetova, Leading researcher. H-index: 4. ORCID ID: 0000-0002-2806-3009.
- Alena Alexandrova, Senior researcher. H-index: 3. ORCID ID: 0000-0002-6501-3510. Scopus author ID: 57195066389. Researcher ID: Q-1176-2017.
- Leila Nadirova, H-index: 1. Scopus ID: 57190008340.
- Rufina Nargilova, H-index: 1. ORCID ID: 0000-0002-9787-8981.
- Evgeniy Shreiber,
- Irina Zhienova,laboratory assistant.
Inventory numbers of interim and final reports:
0322РК01179 (2022), 0323РК00234 (2023), 0224РК00124 (2024).