AP14869357 “Development of the scientific basis of biotechnology for increasing plant productivity through modification of the genes of the ribosomal protein S6”

Project: AP14869357 “Development of the scientific basis of biotechnology for increasing plant productivity through modification of the genes of the ribosomal protein S6”

The main idea of the Project

It is planned to clone the cDNAs of two isoforms of ribosomal protein S6 from Arabidopsis thaliana (AtRPS6A and AtRPS6B) and, through in vitro mutagenesis, replace the triplets encoding serines and threonines at the C-terminus of these proteins with a codon of negatively charged glutamic acid. Mutated cDNAs AtRPS6A(S231E,S237E,S240E,S241E,S247E,T249E) and AtRPS6B(S231E,S237E,S240E,S241E) will be inserted into the genome of potato plants as part of a DNA cassette of a binary vector. It is expected that the polypeptides encoded by these cDNA genes, which imitate the phosphorylated form of AtRPS6 proteins, when integrated into the 40S ribosomal subunits, will activate the translation of the 5′TOP-mRNA family and lead to increased ribosome biogenesis and intensification of overall protein biosynthesis. Under favorable growth conditions, this should lead to increased productivity and earlier maturation of plants.

The Project targets

To develop the scientific basis of biotechnology for producing genetically modified (GM) potato plants (Solanum tuberosum) with increased growth and maturation rates based on the expression of phosphomimetically mutated cDNA genes of ribosomal protein S6 from A. thaliana (AtRPS6A and AtRPS6B).

The Project objectives

  1. Клонировать кДНК двух изоформ рибосомного белка S6 из thaliana (AtRPS6A и AtRPS6B) в вектор pET23c. Мутировать триплеты, кодирующие серин и треонин на С-конце белков AtRPS6A и AtRPS6B, на кодон глютаминовой кислоты.
  2. Express mutated cDNAs in bacterial cells and confirm the synthesis of the proteins they encode using immunoblotting. Purify recombinant proteins His-AtRPS6A(S231E,S237E,S240E,S241E,S247E,T249E) and His-AtRPS6B(S231E,S237E,S240E,S241E) by IMAC chromatography.
  3. Insert variants of the AtRPS6A and AtRPS6B cDNA genes with phosphomimetic substitutions into the Agrobacterium binary vector.
  4. Conduct transient expression of transgenes in plants to functionally confirm their ability to be expressed in vivo.
  5. To establish stable transformation of potatoes with constructs carrying mutated cDNA variants AtRPS6A or At Use the PCR method to check for the presence of transgene inserts.
  6. Assess the level of transgene expression in genetically modified potato plants. To compare the biomass gain and growth rate of control and GM potato plants expressing mutated variants of the AtRPS6A or AtRPS6B cDNA genes.
  7. To evaluate the effect of expression of the cDNA genes AtRPS6A(S231E,S237E,S240E,S241E,S247E,T249E) and AtRPS6B(S231E,S237E,S240E,S241E) on the polyribosomal profile in the plant system in vivo.

Publications and methodological developments

No publication’s at this time.

Research group

  1. Andrey Zhigailov, Associate professor, project supervisor, head of the Laboratory of Protein and Nucleic Acids. H-index: 3. ORCID ID: 0000-0002-9646-033X. Scopus ID: 6508121286. Web of science ID: N-6073-2015.
  2. Ruslan Kryldakov, Principal investigator, senior researcher. H-index: 2. ORCID ID: 0000-0002-2299-3109.
  3. Bulat Iskakov, Doctor of biology, professor, chief researcher. H-index: 6. ORCID ID: 0000-0002-5204-4377.
  4. Gulshan Stanbekova, Associate professor, leading researcher. H-index: 5. ORCID ID: 0000-0002-7819-6475.
  5. Oksana Karpova, Leading researcher. H-index: 2. ORCID ID: 0000-0001-9643-2913.
  6. Anna Nizkorodova, Associate professor, leading researcher. H-index: 1. ORCID ID: 0000-0002-1597-7207.
  7. Nailya Polimbetova, Leading researcher. H-index: 3. ORCID ID: 0000-0002-2806-3009.
  8. Alena Alexandrova, Senior researcher. H-index: 1. ORCID ID: 0000-0002-6501-3510. Scopus author ID: 57195066389. Researcher ID: Q-1176-2017.
  9. Leila Nadirova, researcher. H-index: 1. Scopus ID: 57190008340.
  10. Rufina Nargilova, researcher. H-index: 1. ORCID ID: 0000-0002-9787-8981.
  11. Evgeniy Shreiber,
  12. Irina Zhienova, laboratory assistant.
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