E. Dolgikh, Elizaveta S. Kantsurova, P. Kozyulina, Andrej Bovin, A. V. Dolgikh, Alina M. Dymo, Nikolai V. Kozlov
The development of legume-rhizobium symbiosis is based on signal exchange between partners, which leads to the formation of nitrogen-fixing root nodules. Under the influence of rhizobial signal molecules, the Nod factors, the signal transduction cascade is activated, where the LysM-type receptor kinases and a complex of intracellular regulators, a significant part of which are still unknown, can play an important role. Using transcriptomic and proteomic analysis, we searched for new regulators of the signal pathway in pea Pisum sativum L., which are activated under the influence of Nod factors. Phospholipases C and D, GTPases, calcium-dependent protein kinases, and mitogen-activated protein kinases (MAPKs) have been identified among such novel regulators. The influence of one of such regulators, the MAPK6, on the development of symbiosis was studied in more detail. Using genetic engineering approaches, we increased the transcriptional activity of MAPK6 in transgenic roots, which led to an increase in the number of nodules and the biomass of pea plants. A similar effect was also found for the homologous MAPK6 gene in another legume Medicago truncatula, which has a type of nodulation similar to that one in pea. New approaches have been developed to obtain stable pea transformants with enhanced level of MAPK6 transcription using the constitutive p35S promoter. A comprehensive study of such plants inoculated with rhizobia was carried out. In addition, the approaches for genome editing of pea plants have been designed using the CRISPR/Cas system, when the MAPK6 gene was used as a target. Thus, the possibility of using genetic engineering methods to obtain plants with increased symbiosis efficiency was investigated.
{"title":"Genetically modified legume plants as a basis for studying the signal regulation of symbiosis with nodule bacteria","authors":"E. Dolgikh, Elizaveta S. Kantsurova, P. Kozyulina, Andrej Bovin, A. V. Dolgikh, Alina M. Dymo, Nikolai V. Kozlov","doi":"10.17816/ecogen568623","DOIUrl":"https://doi.org/10.17816/ecogen568623","url":null,"abstract":"The development of legume-rhizobium symbiosis is based on signal exchange between partners, which leads to the formation of nitrogen-fixing root nodules. Under the influence of rhizobial signal molecules, the Nod factors, the signal transduction cascade is activated, where the LysM-type receptor kinases and a complex of intracellular regulators, a significant part of which are still unknown, can play an important role. \u0000Using transcriptomic and proteomic analysis, we searched for new regulators of the signal pathway in pea Pisum sativum L., which are activated under the influence of Nod factors. Phospholipases C and D, GTPases, calcium-dependent protein kinases, and mitogen-activated protein kinases (MAPKs) have been identified among such novel regulators. The influence of one of such regulators, the MAPK6, on the development of symbiosis was studied in more detail. Using genetic engineering approaches, we increased the transcriptional activity of MAPK6 in transgenic roots, which led to an increase in the number of nodules and the biomass of pea plants. A similar effect was also found for the homologous MAPK6 gene in another legume Medicago truncatula, which has a type of nodulation similar to that one in pea. New approaches have been developed to obtain stable pea transformants with enhanced level of MAPK6 transcription using the constitutive p35S promoter. A comprehensive study of such plants inoculated with rhizobia was carried out. In addition, the approaches for genome editing of pea plants have been designed using the CRISPR/Cas system, when the MAPK6 gene was used as a target. Thus, the possibility of using genetic engineering methods to obtain plants with increased symbiosis efficiency was investigated.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"3 9","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138603396","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Anna S. Kiseleva, A. Matveenko, V. Tvorogova, Lyudmila A. Lutova
Legumes are important agricultural and food crops, however, some legume species have a low regenerative capacityin vitro, which complicates obtaining the genetically modified plants with improved properties and analysing gene function. To search for genes that stimulate somatic embryogenesis and to increase the regeneration frequency in legumesin vitro, we designed a screening vector system that will allow faster cloning of genes encoding potential regulators of morphogenesis by preserving restriction sites in the final vector. The construction of vectors is based on the Golden Gate [1] modular cloning method. Using type II restriction endonucleases, DNA fragments form sticky ends and are combined in a given order to form multigene constructs intended forAgrobacterium-mediated transformation. In order to identify efficient variants for gene expression, we used a number of promoters: CaMV 35S(long), CaMV 35S(double), nopaline synthase (nos), actin 2 (act2), and a number of terminators: 35S CaMV, nos, act2 forMtWOX9-1overexpression.Medicago truncatulaWOX9-1 (MtWOX9-1) is a WUSCHEL- related homeobox transcription factor for which a positive effect on the formation of somatic embryos in callus culture was previously shown [2]. Based on the analysis of embryogenic tissue, the optimal combination of promoters and terminators will be selected to assemble the vector for screening of morphogenetic regulators. The work is supported by the Ministry of Science and Higher Education of the Russian Federation (Agreement 075-10-2021-093, Project PBB-RND-2243).
{"title":"The screening vector system of morphogenic regulators in Fabaceae","authors":"Anna S. Kiseleva, A. Matveenko, V. Tvorogova, Lyudmila A. Lutova","doi":"10.17816/ecogen568518","DOIUrl":"https://doi.org/10.17816/ecogen568518","url":null,"abstract":"Legumes are important agricultural and food crops, however, some legume species have a low regenerative capacityin vitro, which complicates obtaining the genetically modified plants with improved properties and analysing gene function. \u0000To search for genes that stimulate somatic embryogenesis and to increase the regeneration frequency in legumesin vitro, we designed a screening vector system that will allow faster cloning of genes encoding potential regulators of morphogenesis by preserving restriction sites in the final vector. The construction of vectors is based on the Golden Gate [1] modular cloning method. Using type II restriction endonucleases, DNA fragments form sticky ends and are combined in a given order to form multigene constructs intended forAgrobacterium-mediated transformation. In order to identify efficient variants for gene expression, we used a number of promoters: CaMV 35S(long), CaMV 35S(double), nopaline synthase (nos), actin 2 (act2), and a number of terminators: 35S CaMV, nos, act2 forMtWOX9-1overexpression.Medicago truncatulaWOX9-1 (MtWOX9-1) is a WUSCHEL- related homeobox transcription factor for which a positive effect on the formation of somatic embryos in callus culture was previously shown [2]. \u0000Based on the analysis of embryogenic tissue, the optimal combination of promoters and terminators will be selected to assemble the vector for screening of morphogenetic regulators. \u0000The work is supported by the Ministry of Science and Higher Education of the Russian Federation (Agreement 075-10-2021-093, Project PBB-RND-2243).","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"2 21","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138603897","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Dmitriy N. Miroshnichenko, V. Timerbaev, M. Divashuk, Aleksandr S. Pushin, Valeria Alekseeva, P. Kroupin, M. Bazhenov, Maria A. Samarina, Aleksey Ermolaev, G. Karlov, Sergey V. Dolgov
The presence of several sets of chromosomes in polyploid crops is a serious problem for the application of gene and genome editing systems. Efficient CRISPR/Cas-based mutagenesis of series of genes involved in the grain starch biosynthesis of hexaploid triticale has been developed. Triticale (×Triticosecale),is a hybrid of rye (Secale) and wheat (Triticum) and consists of three subgenomes. Four genes were targeted and to ensure efficient editing of all subgenomes, a trio of guide RNAs for each target genes were designed. To enable simultaneous editing of 36 genetic loci at once (three sgRNAs × four genes × three subgenomes), an expression cassette was constructed, assembled as an array of twelve sgRNAs. The polysitron vector was delivered to morphogenic calli using a gene gun [1] together with a vector encoding Cas9 nuclease [2] to induce mutations. A number of transgenic plants of spring and winter triticale carrying both Cas9 and sgRNAs inserts have been generated. The efficiency of native gene editing varied depending on the target gene and sgRNA activity. Using a trio of sgRNAs for each target gene, we successfully mutated all three subgenome copies, thereby modifying seed starch synthesis. It can be expected that the described approach will make an important contribution to the future breeding of polyploid crops to produce various combinations of new genetic alleles encoding desired traits. The study is supported by Kurchatov Genomics Center of All-Russia Research Institute of Agricultural Biotechnology, agreement No. 075-15-2019-1667.
{"title":"Advancing gene editing: multiplex mutagenesis in hexaploid triticale","authors":"Dmitriy N. Miroshnichenko, V. Timerbaev, M. Divashuk, Aleksandr S. Pushin, Valeria Alekseeva, P. Kroupin, M. Bazhenov, Maria A. Samarina, Aleksey Ermolaev, G. Karlov, Sergey V. Dolgov","doi":"10.17816/ecogen568624","DOIUrl":"https://doi.org/10.17816/ecogen568624","url":null,"abstract":"The presence of several sets of chromosomes in polyploid crops is a serious problem for the application of gene and genome editing systems. Efficient CRISPR/Cas-based mutagenesis of series of genes involved in the grain starch biosynthesis of hexaploid triticale has been developed. Triticale (×Triticosecale),is a hybrid of rye (Secale) and wheat (Triticum) and consists of three subgenomes. Four genes were targeted and to ensure efficient editing of all subgenomes, a trio of guide RNAs for each target genes were designed. To enable simultaneous editing of 36 genetic loci at once (three sgRNAs × four genes × three subgenomes), an expression cassette was constructed, assembled as an array of twelve sgRNAs. The polysitron vector was delivered to morphogenic calli using a gene gun [1] together with a vector encoding Cas9 nuclease [2] to induce mutations. A number of transgenic plants of spring and winter triticale carrying both Cas9 and sgRNAs inserts have been generated. The efficiency of native gene editing varied depending on the target gene and sgRNA activity. Using a trio of sgRNAs for each target gene, we successfully mutated all three subgenome copies, thereby modifying seed starch synthesis. It can be expected that the described approach will make an important contribution to the future breeding of polyploid crops to produce various combinations of new genetic alleles encoding desired traits. \u0000The study is supported by Kurchatov Genomics Center of All-Russia Research Institute of Agricultural Biotechnology, agreement No. 075-15-2019-1667.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"68 14","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138605052","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Plant somatic cells can be reprogrammed into totipotent embryonic cells that are able to form embryos in a process called somatic embryogenesis (SE). SE can occur naturally in some plant species and it is widely used for the plant’s genetic transformation and regeneration. This process is regulated by hormone treatment and many proteins, among which WUSCHEL-related homeobox (WOX) transcription factors are believed to play crucial roles. Our previous studies have shown thatMtWOX9-1stimulates SE inMedicago truncatula. The aim of the present research was to search for newMtWOXgenes regulating SE. In this study, using transcriptomic data and literature data, we had selected several genes with an increased expression level during SE or in the generative organs and examined the overexpression effect of these genes on the SE ability. It was found that explants of theM.truncatulaembryogenic line, transformed with the construct forMtWOX6-likeoverexpression, develop more somatic embryos compared to the control. Our findings could be a helpful point for searching and studying new morphogenic regulators controlling SE and could have a positive impact on plant biotechnology in improving the transformation and regeneration capacity for other legumes. This work was supported by the Ministry of Science and Higher Education of the Russian Federation in accordance with agreement No. 075-15-2022-322 date 22.04.2022 on providing a grant in the form of subsidies from the Federal budget of Russian Federation. The grant was provided for state support for the creation and development of a World-class Scientific Center “Agrotechnologies for the Future”.
{"title":"The MtWOX genes in the regulation of Medicago truncatula somatic embryogenesis","authors":"Elena P. Efremova, V. Tvorogova, L. Lutova","doi":"10.17816/ecogen568389","DOIUrl":"https://doi.org/10.17816/ecogen568389","url":null,"abstract":"Plant somatic cells can be reprogrammed into totipotent embryonic cells that are able to form embryos in a process called somatic embryogenesis (SE). SE can occur naturally in some plant species and it is widely used for the plant’s genetic transformation and regeneration. \u0000This process is regulated by hormone treatment and many proteins, among which WUSCHEL-related homeobox (WOX) transcription factors are believed to play crucial roles. Our previous studies have shown thatMtWOX9-1stimulates SE inMedicago truncatula. The aim of the present research was to search for newMtWOXgenes regulating SE. In this study, using transcriptomic data and literature data, we had selected several genes with an increased expression level during SE or in the generative organs and examined the overexpression effect of these genes on the SE ability. It was found that explants of theM.truncatulaembryogenic line, transformed with the construct forMtWOX6-likeoverexpression, develop more somatic embryos compared to the control. \u0000Our findings could be a helpful point for searching and studying new morphogenic regulators controlling SE and could have a positive impact on plant biotechnology in improving the transformation and regeneration capacity for other legumes. \u0000This work was supported by the Ministry of Science and Higher Education of the Russian Federation in accordance with agreement No. 075-15-2022-322 date 22.04.2022 on providing a grant in the form of subsidies from the Federal budget of Russian Federation. The grant was provided for state support for the creation and development of a World-class Scientific Center “Agrotechnologies for the Future”.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"21 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138603674","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Naturally transgenic plants are plants that have been subjected to “Agrobacterium” mediated transformation in natural conditions without any human impact. They contain T-DNA-like sequences, called cellular T-DNA (cT-DNA) in their genomes and transfer them from generation to generation [1]. At the moment, several dozen species of natural GMOs are known, and this list is constantly updated. Based on the available data on the diversity of natural GMOs, it can be concluded that in each case, plants have their own set of functionally active transgenes. Accordingly, each cT-DNA performs its own functions. This set of active transgenes will define promising areas for nGMO research, such as: description of the structures and functions of opine synthesis genes and the biological activity of their products in the regulation of plant-microbial interactions [2]; description of the effect of oncogenes on plant morphogenesis, their primary and secondary metabolism [1]. In addition, sequences, newly acquired by plants, can be successfully used in phylogenetic studies [3]. These topics will be the subject of a report at the conference. The work was supported by a grant from the Russian Science Foundation using the equipment of the resource centers of St. Petersburg State University “Biobank” “Chemical Analysis and Materials” and “Development of molecular and cellular technologies”. Keywords: natural GMO; cellular T-DNA; phylogenetic studies; opines; T-DNA oncogenes.
{"title":"Prospects for the study of natural GMOs","authors":"T. Matveeva","doi":"10.17816/ecogen487646","DOIUrl":"https://doi.org/10.17816/ecogen487646","url":null,"abstract":"Naturally transgenic plants are plants that have been subjected to “Agrobacterium” mediated transformation in natural conditions without any human impact. They contain T-DNA-like sequences, called cellular T-DNA (cT-DNA) in their genomes and transfer them from generation to generation [1]. \u0000At the moment, several dozen species of natural GMOs are known, and this list is constantly updated. Based on the available data on the diversity of natural GMOs, it can be concluded that in each case, plants have their own set of functionally active transgenes. Accordingly, each cT-DNA performs its own functions. This set of active transgenes will define promising areas for nGMO research, such as: \u0000 \u0000description of the structures and functions of opine synthesis genes and the biological activity of their products in the regulation of plant-microbial interactions [2]; \u0000description of the effect of oncogenes on plant morphogenesis, their primary and secondary metabolism [1]. \u0000 \u0000In addition, sequences, newly acquired by plants, can be successfully used in phylogenetic studies [3]. \u0000These topics will be the subject of a report at the conference. \u0000The work was supported by a grant from the Russian Science Foundation using the equipment of the resource centers of St. Petersburg State University “Biobank” “Chemical Analysis and Materials” and “Development of molecular and cellular technologies”. \u0000Keywords: natural GMO; cellular T-DNA; phylogenetic studies; opines; T-DNA oncogenes.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"38 5","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138602688","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Today, thanks to use the modern methods of biotechnology, the molecular mechanism underlying different aspects of plant development are started to open up. Along with the traditional methods of genetic analysis, plant developmental genetics actively uses the technics of genetic engineering and “omics” technologies. One of the problems of plant developmental genetics is the study of tumor growth in plants as a model for revealing the mechanisms of systemic control of cell division. Tumor is a pathological structure emerging as a result of uncontrolled proliferation of a group of cells leaving the systemic control of growth rate, cell differentiation and proliferation. Therefore, the elucidation of the mechanisms of tumor formation may help to identify the key regulators of systemic mechanisms controlling cell proliferation and differentiation. Tumor-like structures are found in almost all multicellular organisms, including higher plants. Pathogen-induced tumors, which make up the majority of neoplasms in higher plants, develop under the influence of infectious agents (bacteria, viruses, fungi, nematodes, insects, etc.) which create a niche for their own habitation in the host plant’s organism mostly by shifting the phytohormonal balance and sometimes activation of the meristematic competence of plant cells or modulation of plant cell cycle. At the same time, much rarer spontaneous tumors of higher plants are formed in plants with certain genotypes (mutants, interspecific hybrids, inbred lines) in the absence of any pathogen, which makes them closer to animal tumors. In particular, in the genetic collection of radish (Raphanus sativusL.), the inbred lines that form spontaneous tumors on the taproot during the flowering period were obtained many years ago. The connection between the spontaneous tumor formation in these lines and the altered balance of the main phytohormones, as well as ectopic expression of meristem-specific genes, was previously demonstrated. We have analyzed the differential gene expression in the spontaneous tumors of radish versus the lateral roots using the RNA-seq method. Data were obtained indicating the increased expression of genes associated with cell division and growth (especially genes that regulate G2-M transition and cytokinesis) in the spontaneous tumor. Among genes downregulated in tumor tissue, genes participating in the response to stress and wounding, as well as in the biosynthesis of glucosinolates, were enriched. Subsequently, we also performed whole genome sequencing of two closely related radish lines, contrasting in their ability to spontaneous tumor formation. In the coding genes of tumorous line, we have identified numerous SNPs and InDel which lead to frameshift and are probably associated with the tumor formation trait. Thus, spontaneous tumor formation in inbred lines of radish is probably under complex polygenic control. Testing of the relationship of these polymorphisms with tumor formation has begun.
{"title":"Basic research in the developmental genetics on the model of tumor growth in higher plants","authors":"Lyudmila A. Lutova, Irina E. Dodueva","doi":"10.17816/ecogen568363","DOIUrl":"https://doi.org/10.17816/ecogen568363","url":null,"abstract":"Today, thanks to use the modern methods of biotechnology, the molecular mechanism underlying different aspects of plant development are started to open up. Along with the traditional methods of genetic analysis, plant developmental genetics actively uses the technics of genetic engineering and “omics” technologies. One of the problems of plant developmental genetics is the study of tumor growth in plants as a model for revealing the mechanisms of systemic control of cell division. Tumor is a pathological structure emerging as a result of uncontrolled proliferation of a group of cells leaving the systemic control of growth rate, cell differentiation and proliferation. Therefore, the elucidation of the mechanisms of tumor formation may help to identify the key regulators of systemic mechanisms controlling cell proliferation and differentiation. Tumor-like structures are found in almost all multicellular organisms, including higher plants. Pathogen-induced tumors, which make up the majority of neoplasms in higher plants, develop under the influence of infectious agents (bacteria, viruses, fungi, nematodes, insects, etc.) which create a niche for their own habitation in the host plant’s organism mostly by shifting the phytohormonal balance and sometimes activation of the meristematic competence of plant cells or modulation of plant cell cycle. At the same time, much rarer spontaneous tumors of higher plants are formed in plants with certain genotypes (mutants, interspecific hybrids, inbred lines) in the absence of any pathogen, which makes them closer to animal tumors. In particular, in the genetic collection of radish (Raphanus sativusL.), the inbred lines that form spontaneous tumors on the taproot during the flowering period were obtained many years ago. The connection between the spontaneous tumor formation in these lines and the altered balance of the main phytohormones, as well as ectopic expression of meristem-specific genes, was previously demonstrated. We have analyzed the differential gene expression in the spontaneous tumors of radish versus the lateral roots using the RNA-seq method. Data were obtained indicating the increased expression of genes associated with cell division and growth (especially genes that regulate G2-M transition and cytokinesis) in the spontaneous tumor. Among genes downregulated in tumor tissue, genes participating in the response to stress and wounding, as well as in the biosynthesis of glucosinolates, were enriched. Subsequently, we also performed whole genome sequencing of two closely related radish lines, contrasting in their ability to spontaneous tumor formation. In the coding genes of tumorous line, we have identified numerous SNPs and InDel which lead to frameshift and are probably associated with the tumor formation trait. Thus, spontaneous tumor formation in inbred lines of radish is probably under complex polygenic control. Testing of the relationship of these polymorphisms with tumor formation has begun.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"21 8","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138602994","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Elena S. Okulova, M. Burlakovskiy, M. Padkina, Lyudmila A. Lutova
At the Laboratory of Plant Genetic and Cellular Engineering, Department of Genetics and Biotechnology, St. Petersburg State University, five transgenic Medicago truncatula plants were obtained through Agrobacterium-mediated transformation, carrying one of the variants of the heterologous chicken interferon-gamma gene under the control of the constitutive 35S CaMV promoter. Among these, one plant harbored an unmodified gene insertion, while four had a modified gene with a deletion at the protease recognition site, providing resistance to proteolytic degradation. We demonstrate the application of the SWPOP-PCR “genome walking” method to determine the integration sites of T-DNA into the plant genome, identify the number of insertion copies and their orientation. Analysis of the obtained sequences revealed that only one plant exhibited a single T-DNA insertion, which represents the most optimal structure for stable expression. Upon self-pollination of T0 plants, 39 offspring were obtained and subjected to testing for the presence and expression of the transgene. Among them, six homozygous plants were identified using molecular methods. Quantitative assessment of transgene expression levels showed significant differences among representatives of different lines and among the offspring derived from a single transformed plant. Among the T1 and T2 progeny, the presence of the heterologous interferon protein in plant tissues was confirmed through Western blot analysis. The engineered barrelclover plants hold potential as bioreactors for the production of chicken interferon-gamma for veterinary applications. The use of an edible plant allows eliminating protein extraction and purification procedures, thereby resulting in a noteworthy reduction in production expenses of up to 80%.
{"title":"Obtaining of transgenic barrelclover plants (Medicago truncatula) producing chicken interferon gamma for veterinary use","authors":"Elena S. Okulova, M. Burlakovskiy, M. Padkina, Lyudmila A. Lutova","doi":"10.17816/ecogen567940","DOIUrl":"https://doi.org/10.17816/ecogen567940","url":null,"abstract":"At the Laboratory of Plant Genetic and Cellular Engineering, Department of Genetics and Biotechnology, St. Petersburg State University, five transgenic Medicago truncatula plants were obtained through Agrobacterium-mediated transformation, carrying one of the variants of the heterologous chicken interferon-gamma gene under the control of the constitutive 35S CaMV promoter. Among these, one plant harbored an unmodified gene insertion, while four had a modified gene with a deletion at the protease recognition site, providing resistance to proteolytic degradation. \u0000We demonstrate the application of the SWPOP-PCR “genome walking” method to determine the integration sites of T-DNA into the plant genome, identify the number of insertion copies and their orientation. Analysis of the obtained sequences revealed that only one plant exhibited a single T-DNA insertion, which represents the most optimal structure for stable expression. \u0000Upon self-pollination of T0 plants, 39 offspring were obtained and subjected to testing for the presence and expression of the transgene. Among them, six homozygous plants were identified using molecular methods. Quantitative assessment of transgene expression levels showed significant differences among representatives of different lines and among the offspring derived from a single transformed plant. Among the T1 and T2 progeny, the presence of the heterologous interferon protein in plant tissues was confirmed through Western blot analysis. \u0000The engineered barrelclover plants hold potential as bioreactors for the production of chicken interferon-gamma for veterinary applications. The use of an edible plant allows eliminating protein extraction and purification procedures, thereby resulting in a noteworthy reduction in production expenses of up to 80%.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"12 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138603126","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Andrey R. Shumega, E. Stepchenkova, S. G. Inge-Vechtomov
CRISPR/Cas9-based genome editing systems are widely used for genetic modification of different organisms. One of the drawbacks of CRISPR/Cas9 methods is the non-specific activity of Cas9, which can lead to accumulation of unwanted mutations in the edited genome [1]. Therefore, the development ofin vivomodels for high-throughput analysis of factors influencing the frequency of mutagenesis associated with the use of CRISPR/Cas9 technologies is a relevant task. YeastSaccharomycescerevisiaeis a convenient object for developing such models [2]. Here we represent a yeastin vivomodel that allows us to evaluate the effects of nucleotide sequence of the protospacer adjacent motif (PAM) and the guide RNA (gRNA) on the efficiency of binding between the gRNA/Cas9 complex and the target sequence in the genome. Since the Cas9 activity is lethal in cells lacking a donor sequence for homologous repair of double-strand breaks caused by this endonuclease, in the proposed test-system, the reduced efficiency of transformation by a plasmid encoding Cas9 and various gRNA variants reflects the efficiency of recognition of the target gene by the gRNA/Cas9 complex. To study the influence of different PAM variants, with a consensus of NGG, on CRISPR/Cas9 activity, we obtained four isogenic strains that differ in their PAM sequence (AGG, TGG, CGG, GGG) in the codon 202 of the chromosomal copy of the reporter geneURA3. To evaluate the effect of incomplete matching between gRNA and the target site sequences, we propose using a series of plasmids based on the pML107 vector, encoding Cas9 and one of the 20 possible gRNA variants with single nucleotide substitutions at each of the 20 positions. The results obtained so far indicate the potential of the proposed approach.
{"title":"Evaluation of non-specific CRISPR/Cas9 activity in a yeast model","authors":"Andrey R. Shumega, E. Stepchenkova, S. G. Inge-Vechtomov","doi":"10.17816/ecogen567918","DOIUrl":"https://doi.org/10.17816/ecogen567918","url":null,"abstract":"CRISPR/Cas9-based genome editing systems are widely used for genetic modification of different organisms. One of the drawbacks of CRISPR/Cas9 methods is the non-specific activity of Cas9, which can lead to accumulation of unwanted mutations in the edited genome [1]. Therefore, the development ofin vivomodels for high-throughput analysis of factors influencing the frequency of mutagenesis associated with the use of CRISPR/Cas9 technologies is a relevant task. YeastSaccharomycescerevisiaeis a convenient object for developing such models [2]. \u0000Here we represent a yeastin vivomodel that allows us to evaluate the effects of nucleotide sequence of the protospacer adjacent motif (PAM) and the guide RNA (gRNA) on the efficiency of binding between the gRNA/Cas9 complex and the target sequence in the genome. Since the Cas9 activity is lethal in cells lacking a donor sequence for homologous repair of double-strand breaks caused by this endonuclease, in the proposed test-system, the reduced efficiency of transformation by a plasmid encoding Cas9 and various gRNA variants reflects the efficiency of recognition of the target gene by the gRNA/Cas9 complex. \u0000To study the influence of different PAM variants, with a consensus of NGG, on CRISPR/Cas9 activity, we obtained four isogenic strains that differ in their PAM sequence (AGG, TGG, CGG, GGG) in the codon 202 of the chromosomal copy of the reporter geneURA3. To evaluate the effect of incomplete matching between gRNA and the target site sequences, we propose using a series of plasmids based on the pML107 vector, encoding Cas9 and one of the 20 possible gRNA variants with single nucleotide substitutions at each of the 20 positions. The results obtained so far indicate the potential of the proposed approach.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"21 14","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138603266","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Third international conference “GMO: history, achievements, social and environmental risks” was held on October 3–5, 2023 in Saint Petersburg State University, Saint Petersburg, Russia in mixed format (on-site and on-line). The conference was attended by researchers from Russia, China, Germany, Spain, India, Moldova, Tadjikistan, Belarus. They discussed new genetic engineering methods, promising results and their promotion in society. A lot of interest was attracted to the sections devoted to the genome editing of various organisms and genetic engineering in agriculture. A number of reports aimed to study the fundamental problems of biology and medicine using genetically modified microorganisms and animals. The whole section was traditionally devoted to environmental studies, involving transgenic organisms, natural GMOs that arose without human intervention. Discussion of society’s attitude towards GMOs concluded the conference, where the importance of improvement of GMO diagnostics, as well as closer interaction between biologists and lawyers for improving legislation in the field of GMOs was noted. The conference was held with support of the Ministry of Science and Higher Education of the Russian Federation in accordance with agreement No. 075-15-2022-322 date 22.04.2022 on providing a grant in the form of subsidies from the Federal budget of Russian Federation. The grant was provided for state support for the creation and development of a World-class Scientific Center “Agrotechnologies for the Future”. The conference organizing committee expresses gratitude to the sponsors: companies Helicon and Synthol.
{"title":"Third International Conference “Genetically modified organisms: the history, achievements, social and environmental risks”","authors":"T. Matveeva","doi":"10.17816/ecogen569179","DOIUrl":"https://doi.org/10.17816/ecogen569179","url":null,"abstract":"Third international conference “GMO: history, achievements, social and environmental risks” was held on October 3–5, 2023 in Saint Petersburg State University, Saint Petersburg, Russia in mixed format (on-site and on-line). The conference was attended by researchers from Russia, China, Germany, Spain, India, Moldova, Tadjikistan, Belarus. They discussed new genetic engineering methods, promising results and their promotion in society. A lot of interest was attracted to the sections devoted to the genome editing of various organisms and genetic engineering in agriculture. A number of reports aimed to study the fundamental problems of biology and medicine using genetically modified microorganisms and animals. The whole section was traditionally devoted to environmental studies, involving transgenic organisms, natural GMOs that arose without human intervention. Discussion of society’s attitude towards GMOs concluded the conference, where the importance of improvement of GMO diagnostics, as well as closer interaction between biologists and lawyers for improving legislation in the field of GMOs was noted. \u0000The conference was held with support of the Ministry of Science and Higher Education of the Russian Federation in accordance with agreement No. 075-15-2022-322 date 22.04.2022 on providing a grant in the form of subsidies from the Federal budget of Russian Federation. The grant was provided for state support for the creation and development of a World-class Scientific Center “Agrotechnologies for the Future”. The conference organizing committee expresses gratitude to the sponsors: companies Helicon and Synthol.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"68 13","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138605053","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Amyloid aggregation is a key factor for the development of a series of lethal and incurable diseases, commonly named amyloidoses. The development of various pathologies might be caused by the aggregation of the same protein. This can be due to the ability of any particular protein to adopt several amyloid conformations, specific for the exact disease (Pick’s and Alzheimer’s disease-specific forms of tau protein). How the specific amyloid conformation is formed in each case is not fully understood. In yeast, translation termination factor Sup35 is one of the most extensively studied amyloidogenic proteins. Sup35 aggregation (induction of [PSI+] prion) inactivates the protein and leads to the suppression of nonsense-mutation as the result of read-through. Prionogenic domain of Sup35 protein (Sup35N) has several specific regions: N-terminal QN-rich region (QN), oligopeptide repeats (NR) and C-terminal region (CTN). Sup35 can form various strains of [PSI+] with predominant involvement of different regions of Sup35N into amyloid core thus mimicking disease-specific strains of amyloids described for human amyloidogenic proteins. We implemented the deletions of fragments encoding 1-39 a.a. (QN region) or 75-123/98-123 a.a. (CTN region) intoSUP35gene of yeastSaccharomyces cerevisiae. Then, we induced aggregation of Sup35 protein in the strains carrying mutatedSUP35gene and got the strains possessing [PSIΔ39+], [PSIΔ75–123+], or [PSIΔ98–123+] prion. A set of strains possessing [PSI+] formed by Sup35 protein with various deletions in Sup35N may be convenient model to study disease-specific strains of amyloids formed by human proteins. This research was funded by Russian Science Foundation (grant 20-14-00148-П) and by the St. Petersburg State University (project 94031363).
{"title":"A set of Saccharomyces cerevisiae strains possessing [PSI+] prion formed by Sup35 protein with various deletions in prionogenic domain","authors":"K. Y. Kulichikhin, J. Sopova, A. Rubel","doi":"10.17816/ecogen567848","DOIUrl":"https://doi.org/10.17816/ecogen567848","url":null,"abstract":"Amyloid aggregation is a key factor for the development of a series of lethal and incurable diseases, commonly named amyloidoses. The development of various pathologies might be caused by the aggregation of the same protein. This can be due to the ability of any particular protein to adopt several amyloid conformations, specific for the exact disease (Pick’s and Alzheimer’s disease-specific forms of tau protein). How the specific amyloid conformation is formed in each case is not fully understood. \u0000In yeast, translation termination factor Sup35 is one of the most extensively studied amyloidogenic proteins. Sup35 aggregation (induction of [PSI+] prion) inactivates the protein and leads to the suppression of nonsense-mutation as the result of read-through. \u0000Prionogenic domain of Sup35 protein (Sup35N) has several specific regions: N-terminal QN-rich region (QN), oligopeptide repeats (NR) and C-terminal region (CTN). Sup35 can form various strains of [PSI+] with predominant involvement of different regions of Sup35N into amyloid core thus mimicking disease-specific strains of amyloids described for human amyloidogenic proteins. \u0000We implemented the deletions of fragments encoding 1-39 a.a. (QN region) or 75-123/98-123 a.a. (CTN region) intoSUP35gene of yeastSaccharomyces cerevisiae. Then, we induced aggregation of Sup35 protein in the strains carrying mutatedSUP35gene and got the strains possessing [PSIΔ39+], [PSIΔ75–123+], or [PSIΔ98–123+] prion. A set of strains possessing [PSI+] formed by Sup35 protein with various deletions in Sup35N may be convenient model to study disease-specific strains of amyloids formed by human proteins. \u0000This research was funded by Russian Science Foundation (grant 20-14-00148-П) and by the St. Petersburg State University (project 94031363).","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"52 22","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138602173","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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