Amyloids are protein aggregates characterized by their insolubility in detergents and ability to form fibrils. They are often associated with various diseases, including neurodegenerative disorders, type 2 diabetes and certain forms of cancer. Amyloids also play important roles in bacteria and different physiological processes in both lower and higher eukaryotes. Together with the laboratory of Prof. Y.O. Chernoff we have developed a comprehensive approach for screening new potentially amyloidogenic proteins. This involves using bioinformatics algorithms to predict protein amyloidogenicity and further verifying using a yeast model. We have created a yeast test system specifically designed to study changes in phenotype in genetically modified Saccharomyces cerevisiae strains [1]. This system involves the production of recombinant amyloidogenic proteins fused with reporter proteins Sup35N or YFP. Using yeast assay, we have investigated 22 human proteins that were predicted to be amyloidogenic by ArchCandy algorithm [2]. Currently, additional in vitro biochemical tests are underway with proteins that have shown the potential to form amyloids in yeast models. There are also plans to evaluate the amyloid-forming ability of specific human proteins in mammalian cell cultures. These various approaches appear to be enhancing our comprehension of the impact of amyloid formation in health and disease.
{"title":"Identifying novel amyloid candidates using bioinformatics algorithms and a yeast model approach","authors":"Andrew A. Zelinsky, A. Rubel, Marina V. Ryabinina","doi":"10.17816/ecogen568129","DOIUrl":"https://doi.org/10.17816/ecogen568129","url":null,"abstract":"Amyloids are protein aggregates characterized by their insolubility in detergents and ability to form fibrils. They are often associated with various diseases, including neurodegenerative disorders, type 2 diabetes and certain forms of cancer. Amyloids also play important roles in bacteria and different physiological processes in both lower and higher eukaryotes. \u0000Together with the laboratory of Prof. Y.O. Chernoff we have developed a comprehensive approach for screening new potentially amyloidogenic proteins. This involves using bioinformatics algorithms to predict protein amyloidogenicity and further verifying using a yeast model. We have created a yeast test system specifically designed to study changes in phenotype in genetically modified Saccharomyces cerevisiae strains [1]. This system involves the production of recombinant amyloidogenic proteins fused with reporter proteins Sup35N or YFP. Using yeast assay, we have investigated 22 human proteins that were predicted to be amyloidogenic by ArchCandy algorithm [2]. Currently, additional in vitro biochemical tests are underway with proteins that have shown the potential to form amyloids in yeast models. There are also plans to evaluate the amyloid-forming ability of specific human proteins in mammalian cell cultures. These various approaches appear to be enhancing our comprehension of the impact of amyloid formation in health and disease.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"17 9","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138603414","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}
Two high-protein root cultures of vegetable pea mutants were received [1]. In continuation a PCR analysis of the obtained root cultures genes was carried out according [2] and the amino acid composition of the cultures protein was clarified in a dry product on the AAA 339TM device [3]. Obtained results confirmed the absence of rhizobia contamination of the cultures, which grow steadily on a hormone-free media for 5 years. PCR analysis revealed that fourrolgenesA,B,C,Dwere inserted into the genome of the root culture with genotypeafaftltl, and two —rol Candrol D— in the genome of the root culture with genotypetltl. The protein composition of the obtained cultures was represented by essential and non-essential amino acids and some others. In four inserts culture, the content of essential, ketogenic, proteinogenic and sulfur-containing amino acids prevailed by 1.5–2 times. Two inserts culture has twice as much aspartic acid and proline. Both cultures lacked tryptophan. The number of inserts determines the amino acid composition most likely.
获得了两个蔬菜豌豆突变体的高蛋白根培养物[1]。接着，根据[2]对获得的根培养基因进行PCR分析，并在AAA 339TM装置上用干燥剂澄清培养蛋白的氨基酸组成[3]。获得的结果证实了在无激素培养基上稳定生长5年的培养物没有根瘤菌污染。PCR分析显示，基因型为afaftll的根培养物基因组中插入了4个基因a、B、C、D，基因型为afaftll的根培养物基因组中插入了2个基因Candrol D -。所获得的培养物的蛋白质组成由必需氨基酸和非必需氨基酸以及其他一些氨基酸代表。在4个插入培养中，必需氨基酸、生酮氨基酸、蛋白质氨基酸和含硫氨基酸的含量占主导地位的1.5-2倍。两个插入培养有两倍的天冬氨酸和脯氨酸。两种培养都缺乏色氨酸。插入的数量最有可能决定氨基酸组成。
{"title":"Some biochemical characteristics of the hairy roots of Pisum sativum L. mutants","authors":"O. Timina, O. Timin, Anna Stepanova","doi":"10.17816/ecogen568310","DOIUrl":"https://doi.org/10.17816/ecogen568310","url":null,"abstract":"Two high-protein root cultures of vegetable pea mutants were received [1]. In continuation a PCR analysis of the obtained root cultures genes was carried out according [2] and the amino acid composition of the cultures protein was clarified in a dry product on the AAA 339TM device [3]. Obtained results confirmed the absence of rhizobia contamination of the cultures, which grow steadily on a hormone-free media for 5 years. PCR analysis revealed that fourrolgenesA,B,C,Dwere inserted into the genome of the root culture with genotypeafaftltl, and two —rol Candrol D— in the genome of the root culture with genotypetltl. The protein composition of the obtained cultures was represented by essential and non-essential amino acids and some others. In four inserts culture, the content of essential, ketogenic, proteinogenic and sulfur-containing amino acids prevailed by 1.5–2 times. Two inserts culture has twice as much aspartic acid and proline. Both cultures lacked tryptophan. The number of inserts determines the amino acid composition most likely.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"2 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138603734","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}
Miklhail A. Tsygankov, A. M. Rumyantsev, M. Padkina
Yeast display (DD) is an efficient technology for exposure and fixation of target proteins on the surface of yeast cells by their fusion with cell wall proteins. The scope of application of DD is very wide. It can be used in the study of protein-protein interactions and antibody screening; for the processing of industrial waste, in the processes of bioadsorption of heavy and rare metals, in the production of chemical compounds and biofuels, and in the production of vaccines. DD has a number of advantages over other cell systems. This is due to the fact that yeast, being eukaryotes, unlike bacteria, can carry out various post-translational modifications, correct folding and secretion of eukaryotic proteins. In our work, we compared the effectiveness of different cell wall proteins for exposing target proteins to the surface ofKomagataella phaffiiyeast cells. Two reporter systems were used, based on the eGFP and the beta-galactosidase genes. The most efficient exposure to the surface was provided by the anchor protein ScAGα1p from the yeastSaccharomyces cerevisiae. The genetic constructs obtained in the work can be used for the production of whole-cell biocatalysts. A yeast strainK. phaffiiwas obtained, containing in its genome a construct for the excretion of the Gumboro disease virus antigen protein — VP2. This strain can be used for the production of a vaccine. 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":"Application of yeast display method in biotechnology and agriculture","authors":"Miklhail A. Tsygankov, A. M. Rumyantsev, M. Padkina","doi":"10.17816/ecogen568181","DOIUrl":"https://doi.org/10.17816/ecogen568181","url":null,"abstract":"Yeast display (DD) is an efficient technology for exposure and fixation of target proteins on the surface of yeast cells by their fusion with cell wall proteins. The scope of application of DD is very wide. It can be used in the study of protein-protein interactions and antibody screening; for the processing of industrial waste, in the processes of bioadsorption of heavy and rare metals, in the production of chemical compounds and biofuels, and in the production of vaccines. DD has a number of advantages over other cell systems. This is due to the fact that yeast, being eukaryotes, unlike bacteria, can carry out various post-translational modifications, correct folding and secretion of eukaryotic proteins. \u0000In our work, we compared the effectiveness of different cell wall proteins for exposing target proteins to the surface ofKomagataella phaffiiyeast cells. Two reporter systems were used, based on the eGFP and the beta-galactosidase genes. \u0000The most efficient exposure to the surface was provided by the anchor protein ScAGα1p from the yeastSaccharomyces cerevisiae. The genetic constructs obtained in the work can be used for the production of whole-cell biocatalysts. A yeast strainK. phaffiiwas obtained, containing in its genome a construct for the excretion of the Gumboro disease virus antigen protein — VP2. This strain can be used for the production of a vaccine. \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":"2 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138603958","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}
Genetic engineering is a powerful set of methods used in basic research, biomedicine, and biotechnology. In the molecular biology laboratory, there are established standards in biosafety to protect humans and the environment from unwanted consequences of genetic engineering although internationally agreed, legally binding biosafety standards have not been developed yet. The World Health Organisation as one international actor provides a “Laboratory Biosafety Manual” outlining key biosafety standards and a “Guidance framework for the responsible use of the life sciences” to foster biorisk assessment strategies which could be implemented by national research institutions [1, 2]. Practitioners in both the life sciences and in biotechnology should be trained in comprehensive biorisk assessments which would also further strengthen the implementation of the international agreement on banning biological weapons [3]. Recently, we reported about lessons learned from conducting two iterations of an international interdisciplinary online workshop on responsible conduct in the life sciences [4]. Here, we provide insights from the third workshop and the impact of the lessons learned from this long-standing workshop series on the own work in the molecular biology laboratories of the organisers in respect to biorisk assessments and science communication.
{"title":"Teaching interdisciplinary courses on responsible conduct in the life sciences — implications for biorisk assessments of GMOs","authors":"Mirko Himmel, A. Malygina, Marina S. Dukhinova","doi":"10.17816/ecogen568584","DOIUrl":"https://doi.org/10.17816/ecogen568584","url":null,"abstract":"Genetic engineering is a powerful set of methods used in basic research, biomedicine, and biotechnology. In the molecular biology laboratory, there are established standards in biosafety to protect humans and the environment from unwanted consequences of genetic engineering although internationally agreed, legally binding biosafety standards have not been developed yet. The World Health Organisation as one international actor provides a “Laboratory Biosafety Manual” outlining key biosafety standards and a “Guidance framework for the responsible use of the life sciences” to foster biorisk assessment strategies which could be implemented by national research institutions [1, 2]. Practitioners in both the life sciences and in biotechnology should be trained in comprehensive biorisk assessments which would also further strengthen the implementation of the international agreement on banning biological weapons [3]. Recently, we reported about lessons learned from conducting two iterations of an international interdisciplinary online workshop on responsible conduct in the life sciences [4]. Here, we provide insights from the third workshop and the impact of the lessons learned from this long-standing workshop series on the own work in the molecular biology laboratories of the organisers in respect to biorisk assessments and science communication.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"11 9","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138604378","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}
E. M. Timonova, A. Kiseleva, Alina A. Berezhnaia, Mikhail A. Nesterov, I. Adonina, Aleksey V. Kochetov, Elena A. Salina
CRISPR/Cas technology makes it possible to induce mutations at defined positions. In breeding-oriented research, this opens up exciting opportunities for the targeted improvement of many agricultural crops. Wheat and barley are among the most important cereals in the world. However, the transformation poses a particular challenge for cereals and is strongly genotype dependent. This is because agrobacteria, which is mostly used for delivering the CRISPR/Cas system, have a limited compatibility with these non-host plants. Transformation of wheat is additionally difficult due to the large genome size and polyploidy. Besides obtaining improved genotypes, the object of the current study was to optimize the method of genomic editing based on the CRISPR/Cas system using particle bombardment for non-model varieties of barley and wheat. In barley, we targeted theNudgene that controls hulled/naked phenotype of the grain. Since the regeneration rate remains an issue for the cultivated cultivars, we used the JD633 vector that carries theGRF4-GIF1chimera to increase the efficiency of regeneration. We obtained five T0plants, carrying mutations. In wheat, targetingPpd-1genes that control photoperiod-dependent floral induction results in Cas9-induced mutations in 52 of 210 T0plants. The developed collection of wheat plants with different new alleles ofPpd-D1andPpd-B1genes is being studied for the expression under short day conditions and the effect on the vegetation period. Thus, we have obtained plants of the cultivated varieties of barley and wheat with edited agronomically important genes, using the improved protocols of biolistic transformation. This work was done within the framework of State Assignment Kurchatov Genomic Center of ICG SB RAS (No. 075-15-2019-1662).
{"title":"Modification of agricultural traits in cultivated varieties of barley and wheat","authors":"E. M. Timonova, A. Kiseleva, Alina A. Berezhnaia, Mikhail A. Nesterov, I. Adonina, Aleksey V. Kochetov, Elena A. Salina","doi":"10.17816/ecogen568184","DOIUrl":"https://doi.org/10.17816/ecogen568184","url":null,"abstract":"CRISPR/Cas technology makes it possible to induce mutations at defined positions. In breeding-oriented research, this opens up exciting opportunities for the targeted improvement of many agricultural crops. Wheat and barley are among the most important cereals in the world. However, the transformation poses a particular challenge for cereals and is strongly genotype dependent. This is because agrobacteria, which is mostly used for delivering the CRISPR/Cas system, have a limited compatibility with these non-host plants. Transformation of wheat is additionally difficult due to the large genome size and polyploidy. \u0000Besides obtaining improved genotypes, the object of the current study was to optimize the method of genomic editing based on the CRISPR/Cas system using particle bombardment for non-model varieties of barley and wheat. In barley, we targeted theNudgene that controls hulled/naked phenotype of the grain. Since the regeneration rate remains an issue for the cultivated cultivars, we used the JD633 vector that carries theGRF4-GIF1chimera to increase the efficiency of regeneration. We obtained five T0plants, carrying mutations. In wheat, targetingPpd-1genes that control photoperiod-dependent floral induction results in Cas9-induced mutations in 52 of 210 T0plants. The developed collection of wheat plants with different new alleles ofPpd-D1andPpd-B1genes is being studied for the expression under short day conditions and the effect on the vegetation period. \u0000Thus, we have obtained plants of the cultivated varieties of barley and wheat with edited agronomically important genes, using the improved protocols of biolistic transformation. \u0000This work was done within the framework of State Assignment Kurchatov Genomic Center of ICG SB RAS (No. 075-15-2019-1662).","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"10 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138604386","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}
Pathogenic viruses cause severe tomato losses around the world despite the development of both classical breeding and biotechnological methods. Since replication of phytoviruses involves the interaction between viral components and host plant factors, therefore loss-of-function mutations in the latters can confer viral resistance in plants. There are evidences that eukaryotic translation elongation factor 1 (eEF1) proteins are involved in the replication of some plant viruses. However, the involvement of individual subunits of the eEF1B in the viral cycle is still poorly understood. This work is devoted to the study of the role of the eEF1B factor in the development of tomato virus infection. The contribution of each of the α, β and γ subunits of the eEF1B factor to tomato viral resistance will be determined by CRISPR-Cas9-induced targeted mutagenesis of corresponding gene sequences. As an applied aspect, we expect to find ways to create tomato plants with increased resistance to certain viral diseases. A series of binary vectors contained sequences encoded different RNAs targeting the eEF1B subunit genes was constructed. As a result of Agrobacterium-mediated transformation of tomato, more than 300 independent transgenic lines were obtained. The presence of expression cassettes with functional genes (Cas9 and sgRNAs) was confirmed by PCR. The presence of mutations in target sequences was detected using T7E1 analysis and sequencing. It turned out that the majority of transgenic lines carrying mutations have a chimeric genotype, and mutations of the target genes in the homozygous state were not detected. The propagation of self-pollinated transgenic plants under greenhouse condition and following analyses of target genes to segregate the insertion of foreign DNA and obtain homozygous mutations in eEF1B subunit sequences are in progress.
{"title":"Knockout of the tomato translational elongation factor using CRISPR-Cas9 technology","authors":"Denis Yu. Baranov, Sergey V. Dolgov, V. Timerbaev","doi":"10.17816/ecogen568327","DOIUrl":"https://doi.org/10.17816/ecogen568327","url":null,"abstract":"Pathogenic viruses cause severe tomato losses around the world despite the development of both classical breeding and biotechnological methods. Since replication of phytoviruses involves the interaction between viral components and host plant factors, therefore loss-of-function mutations in the latters can confer viral resistance in plants. There are evidences that eukaryotic translation elongation factor 1 (eEF1) proteins are involved in the replication of some plant viruses. However, the involvement of individual subunits of the eEF1B in the viral cycle is still poorly understood. \u0000This work is devoted to the study of the role of the eEF1B factor in the development of tomato virus infection. The contribution of each of the α, β and γ subunits of the eEF1B factor to tomato viral resistance will be determined by CRISPR-Cas9-induced targeted mutagenesis of corresponding gene sequences. As an applied aspect, we expect to find ways to create tomato plants with increased resistance to certain viral diseases. A series of binary vectors contained sequences encoded different RNAs targeting the eEF1B subunit genes was constructed. As a result of Agrobacterium-mediated transformation of tomato, more than 300 independent transgenic lines were obtained. The presence of expression cassettes with functional genes (Cas9 and sgRNAs) was confirmed by PCR. The presence of mutations in target sequences was detected using T7E1 analysis and sequencing. It turned out that the majority of transgenic lines carrying mutations have a chimeric genotype, and mutations of the target genes in the homozygous state were not detected. The propagation of self-pollinated transgenic plants under greenhouse condition and following analyses of target genes to segregate the insertion of foreign DNA and obtain homozygous mutations in eEF1B subunit sequences are in progress.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"6 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138604663","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}
The presence of foreign DNA is the main obstacle to the application of biotechnological plant varieties. However, transgene-free technologies in the field of genome editing make it possible to overcome this problem. In most countries that already have legislation in this area, plants without foreign DNA do not require field trials and safety tests. The easiest way to avoid integration of transgenes is the delivery of RNP complexes directly into the cell without the use of plasmids. However subsequent selection of edited cells in the absence of a selective marker and plant regeneration are quite difficult. Therefore, traditional genetic constructs are used more often, despite that the elements of the CRISPR system are integrated into the genome. Backcrossing and cross-pollination are used to get rid of unwanted inserts. There are opportunities to accelerate the selection process, such as the Transgene Killer CRISPR system, which ensures the death of plants carrying transgenes in the early embryonic stages [1]. Constructs based on viral replicons integrated into T-DNA are an alternative option. They provide a high level of transient expression of CRISPR elements which are not integrated into the genome. Such vectors were created on the basis of geminiviruses, rhabdoviruses, potexviruses, potyviruses, bunyaviruses [2]. The ability of viruses to move between cells can be both preserved and lost due to the removal of the corresponding proteins. One of the newest approaches is grafting of shoots onto roots expressing Cas and guide RNA [3]. The addition of tRNA-like motifs to the transcripts ensured their mobility and dispersal along the shoot. Heritable edits were observed in the progeny of grafted plants. Thus, for transgene-free editing technologies of plant genomes are rapidly developing, which will accelerate the commercialization of new varieties with economically valuable traits.
{"title":"Transgene-free genome editing of plants","authors":"Elena V. Mikhaylova","doi":"10.17816/ecogen567964","DOIUrl":"https://doi.org/10.17816/ecogen567964","url":null,"abstract":"The presence of foreign DNA is the main obstacle to the application of biotechnological plant varieties. However, transgene-free technologies in the field of genome editing make it possible to overcome this problem. In most countries that already have legislation in this area, plants without foreign DNA do not require field trials and safety tests. \u0000The easiest way to avoid integration of transgenes is the delivery of RNP complexes directly into the cell without the use of plasmids. However subsequent selection of edited cells in the absence of a selective marker and plant regeneration are quite difficult. Therefore, traditional genetic constructs are used more often, despite that the elements of the CRISPR system are integrated into the genome. Backcrossing and cross-pollination are used to get rid of unwanted inserts. There are opportunities to accelerate the selection process, such as the Transgene Killer CRISPR system, which ensures the death of plants carrying transgenes in the early embryonic stages [1]. \u0000Constructs based on viral replicons integrated into T-DNA are an alternative option. They provide a high level of transient expression of CRISPR elements which are not integrated into the genome. Such vectors were created on the basis of geminiviruses, rhabdoviruses, potexviruses, potyviruses, bunyaviruses [2]. The ability of viruses to move between cells can be both preserved and lost due to the removal of the corresponding proteins. \u0000One of the newest approaches is grafting of shoots onto roots expressing Cas and guide RNA [3]. The addition of tRNA-like motifs to the transcripts ensured their mobility and dispersal along the shoot. Heritable edits were observed in the progeny of grafted plants. \u0000Thus, for transgene-free editing technologies of plant genomes are rapidly developing, which will accelerate the commercialization of new varieties with economically valuable traits.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"23 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138601805","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}
Zakhar S. Konstantinov, V. Tvorogova, E. A. Potsenkovskaia, Lyudmila A. Lutova
The success of protocols for the genetic transformation of legumes is limited by their low ability to regenerate. Plant regeneration can occur both along the path of shoot regeneration and their further rooting, and along the path of somatic embryogenesis (SE). SE is similar to zygotic embryogenesis (ZE). It is a method of asexual reproduction, in which the somatic cell, due to its totipotency, switches on the embryogenesis program. SE and ZE involve common participants in transcriptional, hormonal, and epigenetic control. Like many processes in the plant organism, SE is controlled by the activity of various stimulants or repressors. As a result of transcriptional analysis of embryogenic and non-embryogenic calli ofMedicago truncatulaat different stages of development, putative genes-inhibitors of SE were found. Using the Golden Gate system, we created vectors for agrobacterial transformation to overexpress genes of interest in embryogenic calli and evaluate their effect on SE. Overexpression of two genes encoding the transcription factors from WRKY and Homobox-WOX families had a significant inhibitory effect on SE (the average number of somatic embryos per callus decreased). TheMtCLE16,a previously found SE inhibitor from the CLE peptides group, was edited and heterozygous frameshift mutants were obtained. The impact of loss of its function on the SE capacity remains to be analyzed. This research was supported by the Sirius University of Science and Technology, project PBB-RND-2243.
{"title":"The search for inhibitors of somatic embryogenesis in Medicago truncatula","authors":"Zakhar S. Konstantinov, V. Tvorogova, E. A. Potsenkovskaia, Lyudmila A. Lutova","doi":"10.17816/ecogen568377","DOIUrl":"https://doi.org/10.17816/ecogen568377","url":null,"abstract":"The success of protocols for the genetic transformation of legumes is limited by their low ability to regenerate. Plant regeneration can occur both along the path of shoot regeneration and their further rooting, and along the path of somatic embryogenesis (SE). SE is similar to zygotic embryogenesis (ZE). It is a method of asexual reproduction, in which the somatic cell, due to its totipotency, switches on the embryogenesis program. \u0000SE and ZE involve common participants in transcriptional, hormonal, and epigenetic control. Like many processes in the plant organism, SE is controlled by the activity of various stimulants or repressors. \u0000As a result of transcriptional analysis of embryogenic and non-embryogenic calli ofMedicago truncatulaat different stages of development, putative genes-inhibitors of SE were found. Using the Golden Gate system, we created vectors for agrobacterial transformation to overexpress genes of interest in embryogenic calli and evaluate their effect on SE. Overexpression of two genes encoding the transcription factors from WRKY and Homobox-WOX families had a significant inhibitory effect on SE (the average number of somatic embryos per callus decreased). \u0000TheMtCLE16,a previously found SE inhibitor from the CLE peptides group, was edited and heterozygous frameshift mutants were obtained. The impact of loss of its function on the SE capacity remains to be analyzed. \u0000This research was supported by the Sirius University of Science and Technology, project PBB-RND-2243.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"38 26","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138602405","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}
CRISPR/Cas systems are presently the most attractive genome editing technology, that is widely used for genetic engineering of various crops and industrial microorganisms. Currently, application of the CRISPR/Cas based genome editing promises advances in microalgae biotechnology aimed at boosting the output of biofuels and valuable bioactive compounds. However, algae remain relatively complex objects for genetic manipulation [1]. The main problems are associated with the need of a species-oriented approach when creating a transformation toolbox due to the peculiarities in the structure of membranes and the cell wall of a particular taxon. The proper selection and design of a CRISPR construct is also required due to the possible presence of a powerful silencing system against introduced genetic constructs in the cell. These difficulties explain the low efficiency of microalgae transformation and the meager list of successfully edited species [1, 2]. The first instance of genome editing in microalgae using CRISPR/Cas was reported inChlamydomonas reinhardtiiP.A. Dang [3]. To date, four transformation methods (Agrobacterium-mediated, particle bombardment, glass beads agitation, electroporation) have been successfully used for editing (knock-in and knock-out) theC. reinhardtiigenome with two types of CRISPR constructs (plasmid and ribonucleoprotein). The developed protocols make it possible to achieve high efficiency of genomic editing — for example, in our study it varied from 10.6% to 68.8% [4]. These benefits along with completely sequenced genome, well-studied genetics, accessibility and haplontic life cycle makesC.reinhardtiian outstanding model organism for CRISPR/Cas application in microalgae research [5].
{"title":"CRISPR/Cas based genome editing in microalgae","authors":"Pavel A. Virolainen, Elena M. Chekunova","doi":"10.17816/ecogen568609","DOIUrl":"https://doi.org/10.17816/ecogen568609","url":null,"abstract":"CRISPR/Cas systems are presently the most attractive genome editing technology, that is widely used for genetic engineering of various crops and industrial microorganisms. Currently, application of the CRISPR/Cas based genome editing promises advances in microalgae biotechnology aimed at boosting the output of biofuels and valuable bioactive compounds. However, algae remain relatively complex objects for genetic manipulation [1]. The main problems are associated with the need of a species-oriented approach when creating a transformation toolbox due to the peculiarities in the structure of membranes and the cell wall of a particular taxon. The proper selection and design of a CRISPR construct is also required due to the possible presence of a powerful silencing system against introduced genetic constructs in the cell. These difficulties explain the low efficiency of microalgae transformation and the meager list of successfully edited species [1, 2]. \u0000The first instance of genome editing in microalgae using CRISPR/Cas was reported inChlamydomonas reinhardtiiP.A. Dang [3]. To date, four transformation methods (Agrobacterium-mediated, particle bombardment, glass beads agitation, electroporation) have been successfully used for editing (knock-in and knock-out) theC. reinhardtiigenome with two types of CRISPR constructs (plasmid and ribonucleoprotein). The developed protocols make it possible to achieve high efficiency of genomic editing — for example, in our study it varied from 10.6% to 68.8% [4]. These benefits along with completely sequenced genome, well-studied genetics, accessibility and haplontic life cycle makesC.reinhardtiian outstanding model organism for CRISPR/Cas application in microalgae research [5].","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"28 8","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138602745","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}
Artemy A. Ivanov, Alexander V. Burlakov, Tatiana S. Golubeva
Late blight is a disease affecting economically important crops, which is caused by the oomycete Phytophthora infestans. Mainly, fungicides are used against it, however they may harm the environment when used in large quantities. Spray-induced gene silencing (SIGS) can become an alternative to the classical fungicides in the fight against P. infestans. SIGS involves the treatment of plants with double-stranded RNA (dsRNA) which triggers the RNA interference mechanism to suppress translation of the target gene. So, it is possible to suppress the expression of genes. We have chosen two P. infestans genes, inf1 and inf4 involved in the different stages of the parasite development. For the production of dsRNA in E. coli HT115 two expression vectors were constructed on the basis of the L4440 plasmid, each carrying a cDNA fragment of these genes between two T7 phage promoters oriented in the opposition to each other. To evaluate the protective effect of the dsRNA potato explants were treated with a solution containing dsRNA of one of the genes or their combination. 24 hours later, phytophthora zoospores were inoculated with the registration of the effect after 5 days. According to the measurement results, the lesion area was significantly larger in plants treated with water than in the other three experimental groups that were treated with dsRNA.
{"title":"Approaches for the protection of Solanum tuberosum from late blight through the regulation of inf1 and inf4 elicitin genes","authors":"Artemy A. Ivanov, Alexander V. Burlakov, Tatiana S. Golubeva","doi":"10.17816/ecogen568381","DOIUrl":"https://doi.org/10.17816/ecogen568381","url":null,"abstract":"Late blight is a disease affecting economically important crops, which is caused by the oomycete Phytophthora infestans. Mainly, fungicides are used against it, however they may harm the environment when used in large quantities. Spray-induced gene silencing (SIGS) can become an alternative to the classical fungicides in the fight against P. infestans. SIGS involves the treatment of plants with double-stranded RNA (dsRNA) which triggers the RNA interference mechanism to suppress translation of the target gene. So, it is possible to suppress the expression of genes. \u0000We have chosen two P. infestans genes, inf1 and inf4 involved in the different stages of the parasite development. For the production of dsRNA in E. coli HT115 two expression vectors were constructed on the basis of the L4440 plasmid, each carrying a cDNA fragment of these genes between two T7 phage promoters oriented in the opposition to each other. \u0000To evaluate the protective effect of the dsRNA potato explants were treated with a solution containing dsRNA of one of the genes or their combination. 24 hours later, phytophthora zoospores were inoculated with the registration of the effect after 5 days. \u0000According to the measurement results, the lesion area was significantly larger in plants treated with water than in the other three experimental groups that were treated with dsRNA.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"20 7","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138602792","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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