Жасмонаты участвуют в регуляции защитных механизмов в растениях при неблагоприятных условиях среды, а также их роста и развития. В основном, знания о путях биосинтеза и передачи сигналов жасмонатов получены благодаря исследованию модельного двудольного растения Arabidopsis thaliana, однако их функции и молекулярные механизмы действия у однодольных растений, а именно у пшеницы, до сих пор остаются малоизученными. В данной работе мы исследовали устойчивость к стрессовым воздействиям мягкой пшеницы сорта Саратовская-60 и созданной на ее основе трансгенной линии (Tr-3) со сверхэкспрессией гена биосинтеза жасмонатов, 12-оксофитодиеноатредуктазы AtOPR3 (12-OXOPHYTODIENOATE REDUCTASE 3) из Arabidopsis thaliana. Несмотря на высокий уровень экспрессии перенесенного гена AtOPR3, содержание жасмоновой кислоты и ее конъюгата с изолейцином оказалось неизменным в листьях интактных трансгенных растений. После механического повреждения листьев, содержание жасмоновой кислоты в изучаемой трансгенной линии Tr-3 оказалось даже ниже в сравнении с поврежденными нетрансгенными растениями пшеницы Саратовская-60. Различий в содержании 12-оксофитодиеновой кислоты и жасмоноил-изолейцина не было. При этом стресс-индуцированный уровень экспрессии собственных генов алленоксидсинтазы, регулируемых жасмонатами, в трансгенных растениях Tr-3 стал выше, в сравнении с нетрансгенным контролем. Трансгенные растения пшеницы проявили повышенную устойчивость к заражению некротрофным грибом Botrytis cinerea, а также к осмотическому стрессу, вызванному полиэтиленгликолем при прорастании семян. В листьях трансгенной линии Tr-3, зараженных B. сinerea, наблюдалась более высокая активность каталазы, фермента антиоксидантной системы, в сравнении с зараженными листьями нетрансгенных растений, что свидетельствует о ее возможной роли в повышении устойчивости Tr-3 к фитопатогену. Таким образом, немногочисленные данные об особенностях функционирования жасмонатной системы в пшенице дополнены новыми данными о роли экспрессии одного из ключевых генов биосинтеза жасмонатов, а именно 12-OXOPHYTODIENOATE REDUCTASE, в регуляции защитных ответов при пониженном стресс-индуцированном уровне жасмоновой кислоты.

Jasmonates are involved in the regulation of protective mechanisms of plants against unfavorable environments as well as in control of their growth and development. Main data on jasmonate biosynthesis and signals transduction pathways were obtained on model dicotyledonous plant Arabidopsis thaliana. Meanwhile, functions and molecular mode of action of these compounds are still poorly investigated in monocotyledons, including wheat. In the present study, the stress tolerance of transgenic line (Tr-3) and nontransgenic plants of bread wheat cv. Saratovskaya-60 was studied. Transgenic line Tr-3 overexpresses AtOPR3 (12-OXOPHYTODIENOATE REDUCTASE 3) gene from A. thaliana encoding the jasmonate biosynthesis enzyme 12-oxophytodienoate reductase. In spite of the high expression level of AtOPR3, the content of jasmonic acid and its conjugate with isoleucine in intact leaves of the transgenic plants remained unchanged. Furthermore, in the mechanically wounded leaves, the content of jasmonic acid in the transgenic line Tr-3 was even lower than in nontransgenic Saratovskaya-60, while the levels of 12-oxophytodienoic acid and jasmonoil-isoleucine did not differ. In the transgenic plants, the stress-induced expression of the endogenous jasmotate-regulated allene oxide synthase gene was, however, higher than in the nontransgenic control. The transgenic wheat plants were more tolerant to infection by necrotrophic fungus Botrytis cinerea and to osmotic stress caused by polyethylene glycol applied to germinating seeds. The Botrytis-inoculated leaves of the transgenic line Tr-3 manifested higher activity of antioxidant enzyme catalase than inoculated leaves of nontransgenic plants. This fact points to the possible role of catalase in the transgene-associated tolerance to the pathogen. Therefore, the available scarce information concerning the peculiarities of functions of the jasmonate system in wheat plants has been amended with the new data on the role of expression of one of the key genes of jasmonate biosynthesis, namely, 12-OXOPHYTODIENOATE REDUCTASE, in control of defense responses even though the stress-induced level of jasmonic acid is decreased.

Среди разновидностей твердой пшеницы, полба (Triticum dicoccum) занимает особое место как ценнейшая крупяная культура, потребность в которой растет в России с каждым годом. Приоритетный интерес наших исследований - применение современных биотехнологических методов, а именно геномного редактирования, для повышения качества зерна пшеницы.

In modern horticulture Plum pox virus (PPV) imposes serious threats to commercial plantations of a wide range of fruit species belonging to genera Prunus. Given the lack of natural genetic resources, which display reliable resistance to PPV infection, there has been considerable interest in using genetic engineering methods for targeted genome modification of stone fruit trees to control Sharka disease caused by PPV. Among the many virus defense mechanisms, RNA interference is shown to be the most promising transgenic disease-control strategy in plant biotechnology. The present study describes the production of transgenic PPV resistant European plum `Startovaya` (P. domestica L.) through the Agrobacterium-mediated transformation of in vitro leaf explants. Due to organogenesis from leaves, the established protocol allows the genetic engineering of the plum genome without losing clonal fidelity of original cultivar. Seven independent transgenic plum lines containing the self-complementary fragments of PPV-CP gene sequence separated by a PDK intron were generated using hpt as a selective gene and uidA as a reporter gene. The transformation was verified through the histochemical staining for β-glucuronidase activity, PCR amplification of appropriate vector products from isolated genomic DNA and Southern blot analysis of hairpin PPV-CP gene fragments. To clarify the virus resistance, plum buds infected by PPV-M strain were grafted onto 1-year-old transgenic plants, which further were grown into mature trees in the greenhouse. As evaluated by RT-PCR, DAS-ELISA, Western blot, Immuno Strip test, and visual observations, GM plum trees remained uninfected over 9 years. Infected branches that developed from grafted buds displayed obvious symptoms of Sharka disease over the years and maintained the high level of virus accumulation, whereby host transgenic trees had been constantly challenged with the pathogen. Since the virus was unable to spread to transgenic tissues, the stable expression of PPV-derived gene

For the form of dwarf winter-hardy clonal rootstocks of stone crops 1 46-2 ( Prunus pumila L.x P.tomentosa Thunb . ), system of regeneration and genetic transformation using green fluorescent protein (GFP) has been developed. For eff ective regeneration of accessory shoots, no pre-treatment with 6-benzylamine-purine (BA) and auxin was required. Stimulation of the regeneration of shoots from leaf explants required 2-3 weeks of a dark period. Th e best percentage of regeneration (greater than 75 %) was observed with a combination of 3 mg/L BA and 0.75 mg/L IBA. The achieved regeneration effi ciency made it possible to develop a protocol for genetic transformation, mediated by Agrobacterium , for rootstock 146-2. Whole leaves from in vitro-cultured shoots were used as explants for transformation by the A. tumefaciens strain CBE21, with the binary vector pBINmGFP5ER containing the nptII encoding neomycin phosphotransferase II as a plant-selectable marker under the control of the NOS promoter (nopalin synthase) and the reporter gfp gene encoding a green fluorescent protein under the control of the cauliflower mosaic virus (CaMV) promoter 35S. Th e integration of nptII and gfp into transgenes was confirmed by PCR. Expression of the green fluorescent protein was observed using fluorescence microscopy. The efficiency of transformation based on PCR analysis of independent lines resistant to kanamycin was 0.41-0.83 %. All transgenic lines showed resistance to kanamycin at a concentration of 40 mg/L. They were rooted and acclimatized to greenhouse conditions. Th e developed protocols will be used to produce Plum pox virus (PPV) resistant plants.

The method of RNA interference gene expression silencing was used to obtain Plum pox virus (PPV) resistant rootstock and commercial variety Startovaya.For this purpose, a vector with self-complementary sequences of the 578 bp eIF(iso)4G and eIF(iso)E genes fragment was created. The eIF(iso)4G and eIF(iso)E genes encodes factors of initiation of translation involved in the life cycle of a Plum pox virus. A strong promoter of the ribulose-1.5-bisphosphate carboxylase/oxygenase (RuBisCo) gene was chosen to drive the expression of RNA interference hairpin in full and truncated variants. Successful genetic transformation of the 146-2 rootstock and variety Startovaya were carried out by A. tumefaciens CBE21 strain. Whole leaves from in vitro cultured shoots were used as an explant source. The nptII and hpt genes coding for neomycin II and hygromycin phosphotransferase were used as a plant-selectable markers. In our experiments, 5 independent transgenic lines of clonal rootstock and variety were obtained and acclimatized to greenhouse conditions. Th eir status was confirmed by PCR and Southern blot analyses. The transformation efficiency was 0.3-0.4 %. One of these lines was grafted with PPV-infected plum buds and its resistance was verified by ELISA. The use of a full-length gene promoter of the small subunit of ribulosobiephosphate carboxylase (RBCS) in the transformation of plants of the Starter variety led to a decrease in plant viability in the case of suppression of the eIF(iso)4E gene and ensured stability at least in the first year after inoculation in the case of suppression of the eIF(iso)4G gene.

We developed an efficient system for agro-bacterial transformation of plum ( Prunus domestica L.) leaf explants using the PMI/mannose and GFP selection system. Th e variety `Startovaya` was transformed using Agrobacterium tumefaciens strain CBE21 carrying the vector pNOV35SGFP. Leaf explants were placed onto a nutrient medium containing various concentrations and combinations of mannose and sucrose to develop an efficient selection system. Nine independent transgenic lines of plum plants were obtained on a regeneration medium containing 20 g/L sucrose and 15 g/L mannose. The highest transformation frequency (1.40 %) was produced using a delayed selection strategy. Starting from the 1st days after transformation and ending by regeneration of shoots from the transgenic callus, selection of transgenic cells was monitored by GFP fluorescence that allowed avoid ing formation of escapes. Integration of the manA and gfp transgenes was confi rmed by PCR and Southern blotting. On the whole, no direct correlation between the fluorescence level and the copy numbers of the transgenes was found in the present study, though the most intensive fluorescence was observed in line #9 with a single-copy insert. The difference of GFP expression level may have been caused by the integration site or by other factors such as DNA methylation and varying copy number. The described transformation protocol using a positive PMI/mannose system is an alternative selection system for production of transgenic plum plants without genes of antibiotic and herbicide resistance, and the use of leaf explants enables retention of variety traits of plum plants.

In stone fruit trees, resistance to Plum pox virus (PPV) can be achieved through the specific degradation of viral RNA by the mechanism of RNA interference (RNAi). Transgenic virus-resistant plants, however, raise serious biosafety concerns due to the insertion and expression of hairpin constructs that usually contain various selective foreign genes. Since a mature stone tree represents a combination of scion and rootstock, grafting commercial varieties onto transgenic virus-tolerant rootstocks is a possible approach to mitigate biosafety problems. The present study was aimed at answering the following question: To what extent are molecular RNAi silencing signals transmitted across graft junctions in transgrafted plum trees and how much does it affect PPV resistance in genetically modified (GM)/non-transgenic (NT) counterparts? Two combinations, NT:GM and GM:NT (scion:rootstock), were studied, with an emphasis on the first transgrafting scenario. Viral inoculation was carried out on either the scion or the rootstock. The interspecific rootstock `Elita` [(Prunus pumila L..P. salicina Lindl.)x(P. cerasifera Ehrh.)] was combined with cv. Startovaya (Prunus domestica L.) as a scion. Transgenic plum lines of both cultivars were transformed with a PPV-coat protein (CP)-derived intron-separate hairpin-RNA construct and displayed substantial viral resistance. High-throughput sequence data of small RNA (sRNA) pools indicated that the accumulation of construct-specific small interfering RNA (siRNA) in transgenic plum rootstock reached over 2 %. The elevated siRNA level enabled the resistance to PPV and blocked the movement of the virus through the GM tissues into the NT partner when the transgenic tissues were inoculated. At the same time, the mobile siRNA signal was not moved from the GM rootstock to the target NT tissue to a level sufficient to trigger silencing of PPV transcripts and provide reliable viral resistance. Th e lack of mobility of transgenederived siRNA molecules was accompanied by the transfer of various endogenous rootstock-specific siRNAs into the NT scion, indicating the exceptional transitivity failure of the studied RNAi signal. The results presented here indicate that transgrafting in woody fruit trees remains an unpredictable practice and needs further in-depth examination to deliver molecular silencing signals.

The method of RNA interference gene expression silencing was used to obtain plum pox virus (PPV) resistant rootstock plants. For this, a vector with self-complementary sequences of the 578 bp eIF(iso)4G gene fragment was created. The eIF(iso)4G gene encodes a translation initiation factor involved in the life cycle of a Sharka virus. A strong promoter of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) gene was chosen to drive the expression of RNA interference hairpin. Successful genetic transformation of the 146-2 Russian rootstock for plum and apricot was carried out by A. tumefaciens CBE21 strain. Whole leaves from in vitro cultured shoots were used as an explant source. The nptII gene coding for neomycin phosphotransferase II was exploited as a plant-selectable marker. In our experiments, 5 independent transgenic lines were obtained and acclimatized to greenhouse conditions. Their status was confirmed by PCR and Southern blot analyses. The transformation efficiency was 0.3-0.4%. One of these lines was grafted with PPV-infected plum buds and its resistance was verified by ELISA.

Artemisinin-based medicines are the most effective treatment for malaria. To date, the wormwood plants (Artemisia annua L.) are the main source of artemisinin. Due to the limited nature of this source, considerable efforts are directed towards the development of methods for artemisinin production via heterologous expression systems. We used in this study agrobacterial transformation to transfer the genetic module of the artemisinin biosynthesis pathway into plants and then analyzed its transcription in a heterologous host. Tobacco plants were transformed with the artemisinin biosynthesis genes encoding amorpha-4,11-diene synthase, artemisin-aldehyde All(13) reductase, amorpha-4,11-diene monooxygenase, cytochrome P450 reductase from A. annua and yeast 3-hydroxy-3-methylglutaryl-coenzyme A reductase cloned in the pArtemC vector; farnesyl diphosphate synthase and aldehyde dehydrogenase were used to transform the plants as parts of vector p2356. As a result of transformation with the pArtemC and p2356 vectors, in twos transgenic lines with all target genes were obtained. Five genes of artemisinin biosynthesis and two genes of biosynthesis of its precursors were successfully transferred into the genome of transgenic tobacco lines as a result of the co-transformation with abovementioned vectors. Thus, the entire artemisinin biosynthesis pathway was first reconstructed in heterologous plants: the transcription of the artemisinin biosynthesis genes in the tobacco plants was shown via RT-PCR. The obtained results will be used in further research on expression systems for the production of artemisinin and other non-protein substances in heterologous host plants. artemisinin, malaria, metabolic engineering, tobacco, transgenic plants This work was supported by a Grant from the Russian Science Foundation no. 19-14-00190.

A protocol for Agrobacterium-mediated genetic transformation was developed for 146-2 Russian apricot rootstock. Whole leaves from in vitro cultured shoots were used as explants for transformation by A. tumefaciens CBE21 strain containing binary vector pBINmGFP5ER. It has the nptII gene coding for neomycin phosphotransferase II as a plant-selectable marker under the control of the NOS (nopaline synthase) promoter and gfp reporter gene coding the green fluorescent protein under the control of the Cauliflower mosaic virus (CaMV) 35S promoter. Integration of the nptII and gfp into transgenes was confirmed by PCR. The expression of the green fluorescent protein was observed by fluorescence microscopy. Transformation efficiency based on PCR analysis of kanamycin resistant independent lines was 0.41-0.83%. All transgenic lines showed kanamycin resistance at concentration 40 mg L-1. They were rooted and acclimatized to greenhouse conditions.

The present study describes an Agrobacterium-mediated transformation protocol for plum rootstock 'Elita' ((Prunus pumila L. × P. salicina Lindl.) × (P. cerasifera Ehrh.)) using leaf explants. The system enables an efficient plant regeneration with the retention of the traits of the original rootstock. The transformation experiments were conducted using a genetic construct containing the self-complementary sequences of a fragment of PPV-CP gene separated by an intron for inducing Plum pox virus (PPV) resistance through the mechanism of post-transcriptional gene silencing. Transgenic plum plants' rootstocks were produced from organogenic callus developed from leaf explants through a 6-month period culture after inoculation. PCR-analysis confirmed the transgenic status of the produced plants by amplification of the fragments of “hairpin”-PPV-CP construct and hpt gene. To our knowledge, this is the first report of a successful attempt to produce transgenic plum rootstock.

RNA interference strategy was employed for silencing of one of the key genes of the ethylene biosynthesis pathway - apple 1-aminocyclopropane-1-carboxylate (ACC) oxidase in order to prolong shelf life of fruit. Plasmid vectors were developed with spliced hairpin RNA (ihpRNA) in both sense-antisense (sa) and antisense-sense (as) orientations of LeACO1 gene exon4 (496 bp) from tomato and fragment of exon2 (152 bp) of MdACO1 gene from apple driven by constitutive Cauliflower mosaic virus 35S promoter and truncated fruit-specific tomato polygalacturonase promoter (PG). Previously, it has been shown that uidA gene under this promoter is expressed only in ripening fruits of tomato. 62 transformants of an apple hybrid from the Central Genetic Laboratory (Michurinsk, Russia) with marker genes (nptII, hpt) were obtained. Resulting plants were analyzed for the presence of ACO gene fragments. Transgenic plants with confirmed insertion of fragments of ACO genes were grown in greenhouse and grafted on dwarf clonal rootstock N62-396 for earlier fruiting. First part of transgenes was planted in field on certified field plot near Orel city.

Thaumatin II is a supersweet protein derived from the West-African plant Thaumatococcus daniellii Benth. It is a perspective low-calorie sugar substitute for food and pharmaceutical industries. Because of the limitations of its natural sources, obtaining recombinant thaumatin using plant-based expression systems is a promising field of research. This review summarizes many years of research focusing on the physicochemical properties of thaumatins I and II, their roles in plants as pathogenesis-related proteins, and the specific characteristics of their taste perception. A special attention is paid to the detailed description of the studies on obtaining transgenic plants that have been transformed with thaumatin II gene in order to improve their agronomic and consumer properties as well as to obtain recombinant thaumatin for industrial use. Further directions of the research focusing on such areas as obtaining transgenic plants to produce recombinant thaumatin and developing the technologies for its isolation and purification are discussed.

The M2e peptide of avian influenza virus H5N1 Curgan 2005 has successfully been expressed in transgenic tobacco plants. The nucleotide sequence encoding the amino-terminal fragment of the protein M2, 30 a.a. in size that included the M2e peptide (M130) was translationally fused within the reading frame with the 3'-terminus of the ricin B subunit (RTB) sequence in vector pBI121. Tobacco plants were successfully transformed with the obtained construct. The RTB was detected in 4 lines of the transgenic plants from the 8 studied using asialofetuin-immobilized ELISA. The presence of the M2e peptide in the fused protein RTB-M130 was confirmed in two lines of the transgenic plants; in 2 other lines M2e was not detected regardless of the presence of RTB. The quantitative asialofetuin-immobilized ELISA permitted to show that the accumulation of the fused protein RTB-M130 in those lines was equal to 3.3 and 2.4 ^g per 1 gof leaf fresh weight (0.02% and 0.01% of total soluble protein, respectively). The lines in which the expression of the RTB gene was only detected were characterized by the RTB accumulation of 2.1 and 2.4 ^g per 1 gof fresh weight (0.01 and 0.02% of the total soluble protein, respectively). Western blot analysis showed that the RTB-M130 protein was detected in the transgenic tobacco lines as a dimer without significant signs of degradation. The obtained results will be further used for the design of a plant-based edible vaccine against avian influenza.

In this study, we investigated the possibility of increasing the level of transgene expression using DNA element that can terminate transcription.