Project description
Cloning in the binary vector pCambia2300 under the control of a plant promoter and transcription terminator cDNA gene protein kinase-2 of ribosomal protein S6 from Arabidopsis thaliana (AtRPS6K2), mutated in vitro by replacing triplets encoding serine(S)-296, S-437 and threonine(T )-455 per codon of the negatively charged glutamic acid (E). Such phosphomimetic substitutions activate the AtRPS6K2 kinase, which phosphorylates the RPS6 protein in the 40S ribosomal subunits, which is accompanied by an increase in the processes of ribosome biogenesis, cell growth, and division. The resulting genetic construct will be used for stable transformation of the tobacco plant. There is reason to believe that the expression of the mutated AtRPS6K2 (S296E, S437E, T455E) gene in tobacco plants under favorable conditions will lead to an increase in their biomass.
Project goal:
Using tobacco plants (Nicotiana tabacum) as an example, to develop a biotechnology for obtaining genetically modified (GM) plants with increased biomass and productivity based on optimization of the expression of the mutated AtRPS6K2 (S296E, S437E, T455E) cDNA gene encoding the constitutively activated protein kinase form of the ribosomal protein S6 from Arabidopsis thaliana (AtRPS6K2).
Project objectives:
1) Clone into the agrobacterial vector pCambia2300 a mutated variant of the AtRPS6K2 cDNA gene with codon substitutions S296, S437, and T455 for triplets encoding phosphomimetic glutamic acid (E). Explanation: cDNA gene cloning into an agrobacterial vector allows stable genetic transformation of plants of various species.
“Check-point” result: DNA construct with the verified nucleotide sequence 35S-AtRPS6K2(S296E, S437E, T455E)-NOS_pCAMBIA2300..
2) Perform transient expression of the AtRPS6K2(S296E, S437E, T455E) transgene in tobacco plants ( tabacum) to functionally confirm its ability to be expressed in vivo.
Explanation: this stage is necessary in order to check the ability of the assembled DNA cassette to ensure the expression of the target transgene in plant cells of the model species.
“Check-point” result: agrobacteria line Agrobacterium tumefaciens strain EHA-101 carrying the 35S-AtRPS6K2(S296E, S437E, T455E)-NOS_pCAMBIA2300 plasmid.
3) Establish stable transformation and obtain genetically modified (GM) tobacco plants expressing the AtRPS6K2(S296E, S437E, T455E)
Explanation: stable transformation of plants is carried out to transfer the genetic cassette into their genome. It is important that the transgene insert contains all functional segments required for expression: transgene, promoter, and transcription terminator.
“Check-point” result: regenerated GM tobacco plants containing the AtRPS6K2(S296E, S437E, T455E) transgene in the genome under the control of the 35S-CaMV promoter and the NOS transcription terminator.
4) Compare the growth rates of control and GM tobacco plants expressing the AtRPS6K2(S296E, S437E, T455E)
Explanation: only statistically significant differences between GM and control plants in growth rates can prove the effectiveness of the chosen approach to increase plant productivity.
“Check-point: result: lines of GM tobacco plants expressing the AtRPS6K2(S296E, S437E, T455E) transgene, characterized by increased productivity.
Tasks for 2020 and obtained scientific results:
Schedule section: No. 1.1. “Clone the in vitro mutated AtRPS6K2 cDNA gene (S296E, S437E, T455E) into the pCAMBIA2300 vector under the control of the 35S-CaMV promoter and NOS transcription terminator”.
Completed works:
A computer analysis of the nucleotide sequences of AtRPS6K2 mRNA and TEV genomic RNA was carried out. Primers were selected for cloning the AtRPS6K2(S296E, S437E, T455E) cDNA and the 5′-untranslated TEV gRNA sequence into a binary vector. The previously obtained genetic construct pET19b_His-AtRPS6K2(S296E, S437E, T455E) was cloned from the 5´TEV-GUS plasmid 5′-NTP of TEV gRNA at the XbaI/NcoI sites before the open reading frame. The 5’TEV-His-AtRPS6K2(S296E, S296E, S437E, T455E)” according to the XbaI/SacI sites. Section work completed.
Schedule section: No. 1.2. “Check the correctness of the created genetic construct by PCR analysis and restriction method; verify the introduced nucleotide substitutions by DNA sequencing”.
Completed works:
The correctness of the plasmid pCambia-35S-5’TEV-His-AtRPS6K2(S296E, S437E, T455E)-Nos was verified by PCR analysis using primers to the 35S-CaMV promoter, NOS terminator, TEV enhancer, and to the target cDNA- kinase gene, as well as by the restriction method using restriction endonucleases XbaI and SacI. The nucleotide substitutions introduced into the natural cDNA gene were verified by DNA sequencing using a reverse primer to the NOS terminator. Section work completed.
Tasks for 2021 and obtained scientific results:
Schedule section: No. 2.1. “Obtain agrobacteria carrying plasmid 35S-AtRPS6K2 (S296E, S437E, T455E)-NOS_ pCAMBIA2300 for plant transformation”.
Completed works:
Cells of Agrobacterium strain EHA105 were grown on liquid LB medium, brought into a competent form by treatment with magnesium and calcium salts, then transformed by electroporation with plasmid 35S-AtRPS6K2 (S296E, S437E, T455E)-NOS_pCAMBIA2300. Clones were selected on a medium with kanamycin (50 μg/ml), tetracycline (10 μg/ml), and rifampicin (25 μg/ml). The plasmid was inserted into agrobacteria cells by PCR analysis after total DNA was isolated from agrobacteria cells by the CTAB method. Cells of agrobacteria carrying the indicated plasmid are museumized. Section work completed.
Schedule section: No. 2.2. “Carry out transient expression of the AtRPS6K2(S296E, S437E, T455E) transgene in tobacco plants. RT-PCR method to determine the levels of transgene expression”.
Completed works:
The obtained lines of agrobacteria were used for transgene expression in plants. To deliver agrobacteria into the cells of stomata of tobacco plants, the method of vacuum infiltration of leaf discs was used. Four days after inoculation with agrobacteria, RNA preparations were isolated from leaf discs, treated with DNase I, and then RT-PCR was performed with transgene-specific primers. Total water-soluble extracts from leaf discs were analyzed by immunoblotting. In tobacco plants, the synthesis of mRNA with a recombinant DNA cassette embedded in agrobacteria, as well as the synthesis of the target recombinant protein, was confirmed. The level of transgenic mRNA synthesis correlated with the level of recombinant protein synthesis. Section work completed.
Schedule section: No. 3.1. “Optimize and conduct stable genetic transformation of tobacco plants”.
Completed works:
Stable transformation of tobacco was carried out by agroinfection using agrobacterium A. tumefaciens strain EHA 105 transformed with plasmids pCambia-35S-5’TEV-His-AtRPS6K2(S296E, S437E, T455E)-Nos and 35S-NOS_Cambia2300. In the course of optimizing the process of stable genetic transformation of tobacco, it was decided not to add acetosyringone to agrobacteria to minimize the number of inserts per genome; the content of kanamycin in the nutrient medium for screening regenerants was increased from 25 mg/l to 50 mg/l. All work on the section has been completed.
Schedule section: No. 3.2. “Test the resulting regenerated plants for the presence of a transgenic insert by PCR.”
Total DNA preparations were isolated from all grown regenerants of the experimental and control batches. The isolated DNA samples were tested by PCR with two transgene-specific primers (experimental group) and promoter/terminator-specific primers (control group). Of the 42 shoots from the experimental batch, thirteen plants (31.0%) showed the presence of the transgenic insert. Of the 24 regenerants in the control group, six plants (25.0%) were PCR-positive for the transgenic insert. The differences were not statistically significant (p = 0.8172). All work on the section has been completed.
Publications, methodological developments and implementation of results
- A scientific article has been published in a foreign publication that is included in the 1st, 2nd or 3rd quartile of the Web of Science database and (or) has a Scopus percentile of at least 50 (on account of paragraph 4.2 of the Calendar Plan) indicating the IRN of the project and the source of funding (KN MES RK) in the “Financing” section: Stanbekova G., Beisenov D., Nizkorodova A., Iskakov B., Warzecha H. Production of the sheep pox virus structural protein SPPV117 in tobacco chloroplasts // Biotechnology letters. – 2021. – Vol.43, No.7. – P. 1475–1485. https://doi.org/10.1007/s10529-021-03117-x. Percentile (Scopus) — 56%; quartile (WoS) – Q3; IF – 1.977; SiteScore – 3,8; Sjr – 0,548.
- As part of the project, a laboratory regulation “Method for obtaining genetically modified tobacco plants (Nicotiana tabacum) with increased productivity in optimal conditions for growth and their identification by PCR” was developed and formalized accordingly. The technology has been tested in the Republican State Enterprise “Institute of General Genetics and Cytology” of the Committee of Science of the Ministry of Education and Science of the Republic of Kazakhstan and the LLP “Kazakh Research Institute of Potato and Vegetable Growing” (there is an act signed and certified by both of these organizations).
Project executors:
- Zhigailov Andrey Viktorovich, D., Project supervisor, Leading researcher. Hirsch index: 2. Total – 62 citations. ORCID: https://orcid.org/0000-0002-9646-033X. Scopus ID: 6508121286. Web of Science ID: N-6073-2015.
- Kryldakov Ruslan Vladimirovich, Ph.D., Senior r Responsible executive of the project. Hirsch index: 2. ORCID: https://orcid.org/0000-0002-2299-310.
- Iskakov Bulat Kudaibergenovich, Doctor of Biological Sciences, Professor, Head of the Laboratory of protein and nucleic zcids “IMBB”. Hirsch index: 6. Total – 104 citations. ORCID: https://orcid.org/0000-0002-5204-4377. Scopus ID: 6602606343. Web of Science ID: H-5988-2015.
- Polimbetova Nailya Seitzhanovna, Ph.D., Leading r Hirsch index: 2. Total – 46 citations. ORCID: https://orcid.org/0000-0002-2806-3009. Scopus ID: 6506904731. Web of Science ID: N-6709-2015.
- Stanbekova Gulshan Esenbekovna, Ph.D., Leading researcher Hirsch index: 4. In total – 134 citations. ORCID: https://orcid.org/0000-0002-7819-6475. Scopus ID: 6506211551. Web of Science ID: N-6607-2015.
- Karpova Oksana Vladislavovna, Ph.D., Leading r Hirsch index: 2. ORCID: https://orcid.org/0000-0001-9643-2913.
- Nizkorodova Anna Sergeevna, Ph.D., Leading researcher. Hirsch index: 1. Total – 2 citations. ORCID ID: https://orcid.org/0000-0002-1597-7207. Scopus ID: 57215971184. Web of Science ID: AAY-1646-2020.
- Aleksandrova Alena Mikhailovna, PhD-doctoral student, Senior researcher, Hirsch index: 2. Total – 9 citations. ORCID: https://orcid.org/0000-0002-6501-3510. Web of Science ID: 9. I-7557-2018.