Nucleotide sequence of an osmotin cDNA from the Nicotiana tabacum cv. White Burley generated by the polymerase chain reaction
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White (mutation)
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The CLE family (CLAVATA3/embryo surrounding region-related), a class of small secreted proteins, play important roles in plant development and stress responses. Members of the CLE family have been characterized in a number of plant species, including Arabidopsis and rice. However, limited information is available about CLE peptides in tobacco (Nicotiana tabacum) and related Nicotiana species. Here we report the identification of 84 CLE family members in three Nicotiana species based on sequence similarity. The newly identified CLE members, including 41 from N. tabacum, 19 from N. sylvestris, and 24 from N. tomentosiformis, together with 32 CLEs from Arabidopsis and 52 CLEs from tomato, formed 9 subgroups in a phylogenic tree. The unbalanced distribution of the Nicotiana CLEs in the subgroups suggested potential preferential gene family expansion during evolution. Expression of the NtCLE genes was analyzed and a number of the NtCLEs showed induced expression upon abiotic stress treatments. Synthetic peptides of several NtCLEs, when applied to detached tobacco leaf discs, were able to increase plants’ tolerance to osmotic and salinity stresses, suggesting potential roles of CLE peptides in the stress responses of tobacco.
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Sustainable production of chemicals and improving these biosources by engineering metabolic pathways to create efficient plant-based biofactories relies on the knowledge of available chemical/biosynthetic diversity present in the plant. Nicotiana species are well known for their amenability towards transformation and other new plant breeding techniques. The genus Nicotiana is primarily known through Nicotiana tabacum L., the source of tobacco leaves and all respective tobacco products. Due to the prevalence of the latter, N. tabacum and related Nicotiana species are one of the most extensively studied plants. The majority of studies focused solely on N. tabacum or other individual species for chemotyping. The present study analysed a diversity panel including 17 Nicotiana species and six accessions of Nicotiana benthamiana and created a data set that effectively represents the chemotype core collection of the genus Nicotiana. The utilisation of several analytical platforms and previously published libraries/databases enabled the identification and measurement of over 360 metabolites of a wide range of chemical classes as well as thousands of unknowns with dedicated spectral and chromatographic properties.
Chemotype
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Agrobacterium-mediated transformation is the most popular approach for obtaining transgenic plants nowadays. There are plenty of protocols developed for different plant species. These protocols usually include the medium composition, the technology for preparing plant explants and cultivation conditions, as well as the choice of agrobacteria strains. Nicotiana tabacum, or cultivated tobacco, was one of the f irst successfully transformed plant species. Nicotiana tabacum is a model object in plant genetics, particularly due to its ability for transformation and regeneration. N. tabacum is a naturally transgenic plant since its genome contains a cellular T-DNA acquired from Agrobacteria. The signif icance of cT-DNA for plants has not yet been established. Some assume that cT-DNA can increase the ability of plants to regenerate due to some of the genes they contain. For example, rolC has been shown to affect the hormonal balance of plants, but the molecular mechanisms underlying this have yet to be found. RolC is also somehow involved in the secondary metabolism of plants. Like N. tabacum, Nicotiana glauca produces a wide range of secondary metabolites and contains an intact rolC gene in its genome. At the same time, unlike N. tabacum, N. glauca is a diploid species, which makes it more suitable for genetic engineering approaches. Nicotiana sylvestris is one of the ancestral species of N. tabacum and does not contain cT-DNA. The aim of this work was to develop a protocol for transformation and regeneration of N. glauca and N. sylvestris. We managed to find an optimum ratio of auxins and cytokinins that promotes both active callus formation and organogenesis in N. glauca and N. sylvestris leaf explants. The developed technique will be useful both for fundamental research that includes the N. glauca and N. sylvestris species, and for practical application in the pharmaceutical industry and biosynthesis.Агробактериальная трансформация – наиболее популярный метод получения трансгенных растений. Для многих видов растений разработаны протоколы, включающие описание условий трансформации, состав питательных сред, методику подготовки растительных эксплантов и выбор штаммов агробактерий, а также соотношение растительных гормонов, необходимых для последующей регенерации эксплантов. Одним из первых успешно трансформированных видов стал культурный табак, Nicotiana tabacum, который сегодня служит модельным объектом генетики растений. Nicotiana tabacum эффективно трансформируется и легко регенерирует, что делает его удобным для генно-инженерных манипуляций. При этом N. tabacum относится к природно-трансгенным видам, поскольку содержит в своем геноме последовательности агробактериального происхождения, клеточную Т-ДНК, значение которой для растений пока не установлено. Одним из предковых видов для N. tabacum является N. sylvestris, геном которого не содержит клТ-ДНК. Предполагают, что клТ-ДНК может повышать регенерационные способности растения за счет генов, входящих в ее состав, таких как, например, rolC. Для rolC действительно показано влияние на баланс растительных гормонов, однако стоящие за этим молекулярные механизмы остаются неизвестными. Помимо участия в морфогенезе, rolC влияет на биосинтез вторичных метаболитов в растении. Вид N. glauca, как и N. tabacum, считается природно-трансгенным, несет в клТ-ДНК интактный rolC и содержит широкий спектр вторичных метаболитов. При этом, в отличие от N. tabacum, N. glauca –диплоидный вид, что делает его гораздо более удобным объектом для проведения генно-инженерных работ. Целью данной работы была разработка протокола трансформации и регенерации для видов N. glauca и N. sylvestris. На основании уже известных протоколов для других представителей рода Nicotiana нами было подобрано такое соотношение ауксинов и цитокининов, при котором листовые экспланты N. glauca и N. sylvestris переходят к активному каллусообразованию, а затем к органогенезу. С использованием разработанной методики получены трансгенные растения этих видов. Разработанная методика трансформации и регенерации полезна как для фундаментальных исследований, затрагивающих виды N. glauca и N. sylvestris, так и для практического применения в области фарминдустрии и биосинтеза.
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Summary Although many different crop species have been used to produce a wide range of vaccines, antibodies, biopharmaceuticals and industrial enzymes, tobacco has the most established history for the production of recombinant proteins. To further improve the heterologous protein yield of tobacco platforms, transient and stable expression of four recombinant proteins (i.e. human erythropoietin and interleukin‐10, an antibody against Pseudomonas aeruginosa , and a hyperthermostable α‐amylase) was evaluated in numerous species and cultivars of Nicotiana . Whereas the transient level of recombinant protein accumulation varied significantly amongst the different Nicotiana plant hosts, the variety of Nicotiana had little practical impact on the recombinant protein concentration in stable transgenic plants. In addition, this study examined the growth rate, amount of leaf biomass, total soluble protein levels and the alkaloid content of the various Nicotiana varieties to establish the best plant platform for commercial production of recombinant proteins. Of the 52 Nicotiana varieties evaluated, Nicotiana tabacum (cv. I 64) produced the highest transient concentrations of recombinant proteins, in addition to producing a large amount of biomass and a relatively low quantity of alkaloids, probably making it the most effective plant host for recombinant protein production.
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Abstract Somatic hybrid plants were produced by fusion of protoplasts from cell cultures of the Nicotiana tabacum L. sulfur mutant Su/Su and from leaf mesophyll of Nicotiana glauca Graham. After fusion the N. glauca protoplasts failed to survive under the selected culture condition. From the hybrid cells light green shoots were produced. The hybrid plants exhibited intermediate characters between parental species with respect to leaf morphology, trichome density, floral structure and flower color. The chromosome number of 25 hybrid plants was 2n = 72 and both N. glauca and N. tabacum chromosomes were identified in the hybrids. Results of isoenzyme analysis showed bands of both parents and a specific (hybrid) band for aspartate amino‐transferase. Small subunit fraction‐1‐protein of somatic hybrids also consisted of the sum of N. glauca and N. tabacum bands. Leaf spot formation associated with the Su locus of N. tabacum was observed in somatic hybrids.
Somatic fusion
Protoplast
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Проведено исследование 19 диких видов рода Никоциана (Nicotiana) семейство Паслёновые (Solanaceae) коллекции института по химическому составу листьев. Научные исследования выполняли с использованием методик постановки и проведения опытов, разработанных в институте. Выделено 13 диких видов с практическим отсутствием никотина; восемь видов с высоким содержанием углеводов; четыре вида с низким содержанием белков. Эти виды Никоциана могут быть использованы в селекции курительного табака на изменение химического состава листьев. A study of 19 wild species of the genus Nicotiana (family Solanaceae) from the collection of the Institute for the chemical composition of leaves was carried out. Scientific research was carried out using the methods of setting and conducting experiments developed at the Institute. There are 13 wild species identified without nicotine; eight types of high carbohydrate; four types of low protein. These types of Nicotiana can be used in the breeding of smoking tobacco to change the chemical composition of the leaves.
Plant science
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