The dynamics of genome size and GC contents evolution in genus Nicotiana / A dinâmica do tamanho do genoma e a evolução dos conteúdos de GC no gênero Nicotiana

Hybridization and Polyploidization are most common of the phenomenon observed in plants, especially in the genus Nicotiana leading to the duplication of genome. Although genomic changes associated with these events has been studied at various levels but the genome size and GC content variation is less understood because of absence of sufficient genomic data. In this study the flow cytometry technique was used to uncover the genome size and GC contents of 46 Nicotiana species and we compared the genomic changes associated with the hybridization events along evolutionary time scale. The genome size among Nicotiana species varied between 3.28 pg and 11.88 pg whereas GC contents varied between 37.22% and 51.25%. The tetraploid species in genus Nicotiana including section Polydiclae, Repandae, Nicotiana, Rustica and Sauveolentes revealed both up and downsizing in their genome sizes when compared to the sum of genomes of their ancestral species. The genome sizes of three homoploid hybrids were found near their ancestral species. Loss of large genome sequence was observed in the evolutionary more aged species (>10 Myr) as compared to the recently evolved one’s (<0.2 Myr). The GC contents were found homogenous with a mean difference of 2.46% among the Nicotiana species. It is concluded that genome size change appeared in either direction whereas the GC contents were found more homogenous in genus Nicotiana.

A hibridização e a poliploidização são os fenômenos mais comuns observados em plantas, principalmente no gênero Nicotiana, levando à duplicação do genoma. Embora as mudanças genômicas associadas a esses eventos tenham sido estudadas em vários níveis, o tamanho do genoma e a variação do conteúdo de GC são menos compreendidos devido à ausência de dados genômicos suficientes. Neste estudo, a técnica de citometria de fluxo foi usada para descobrir o tamanho do genoma e o conteúdo de GC de 46 espécies de Nicotiana, e comparamos as mudanças genômicas associadas aos eventos de hibridização ao longo da escala de tempo evolutiva. O tamanho do genoma entre as espécies de Nicotiana variou entre 3,28 pg e 11,88 pg, enquanto os conteúdos de GC variaramentre 37,22% e 51,25%. As espécies tetraploides do gênero Nicotiana, incluindo as seções Polydiclae, Repandae, Nicotiana, Rustica e Sauveolentes, revelaram aumento e redução do tamanho do genoma quando comparados à soma dos genomas de suas espécies ancestrais. Os tamanhos do genoma de três híbridos homoploides foram encontrados perto de suas espécies ancestrais. A perda da grande sequência do genoma foi observada nas espécies evolutivas mais velhas (> 10 Myr) em comparação com as que evoluíram recentemente (< 0,2 Myr). Os teores de GC foram homogêneos com diferença média de 2,46% entre as espécies de Nicotiana. Conclui-se que a mudança no tamanho do genoma apareceu em ambas as direções, enquanto os conteúdos de GC foram encontrados mais homogêneos no gênero Nicotiana.

The dynamics of genome size and GC contents evolution in genus Nicotiana

Abstract Hybridization and Polyploidization are most common of the phenomenon observed in plants, especially in the genus Nicotiana leading to the duplication of genome. Although genomic changes associated with these events has been studied at various levels but the genome size and GC content variation is less understood because of absence of sufficient genomic data. In this study the flow cytometry technique was used to uncover the genome size and GC contents of 46 Nicotiana species and we compared the genomic changes associated with the hybridization events along evolutionary time scale. The genome size among Nicotiana species varied between 3.28 pg and 11.88 pg whereas GC contents varied between 37.22% and 51.25%. The tetraploid species in genus Nicotiana including section Polydiclae, Repandae, Nicotiana, Rustica and Sauveolentes revealed both up and downsizing in their genome sizes when compared to the sum of genomes of their ancestral species. The genome sizes of three homoploid hybrids were found near their ancestral species. Loss of large genome sequence was observed in the evolutionary more aged species (>10 Myr) as compared to the recently evolved ones ( 0.2 Myr). The GC contents were found homogenous with a mean difference of 2.46% among the Nicotiana species. It is concluded that genome size change appeared in either direction whereas the GC contents were found more homogenous in genus Nicotiana.

Resumo A hibridização e a poliploidização são os fenômenos mais comuns observados em plantas, principalmente no gênero Nicotiana, levando à duplicação do genoma. Embora as mudanças genômicas associadas a esses eventos tenham sido estudadas em vários níveis, o tamanho do genoma e a variação do conteúdo de GC são menos compreendidos devido à ausência de dados genômicos suficientes. Neste estudo, a técnica de citometria de fluxo foi usada para descobrir o tamanho do genoma e o conteúdo de GC de 46 espécies de Nicotiana, e comparamos as mudanças genômicas associadas aos eventos de hibridização ao longo da escala de tempo evolutiva. O tamanho do genoma entre as espécies de Nicotiana variou entre 3,28 pg e 11,88 pg, enquanto os conteúdos de GC variaram entre 37,22% e 51,25%. As espécies tetraploides do gênero Nicotiana, incluindo as seções Polydiclae, Repandae, Nicotiana, Rustica e Sauveolentes, revelaram aumento e redução do tamanho do genoma quando comparados à soma dos genomas de suas espécies ancestrais. Os tamanhos do genoma de três híbridos homoploides foram encontrados perto de suas espécies ancestrais. A perda da grande sequência do genoma foi observada nas espécies evolutivas mais velhas (> 10 Myr) em comparação com as que evoluíram recentemente ( 0,2 Myr). Os teores de GC foram homogêneos com diferença média de 2,46% entre as espécies de Nicotiana. Conclui-se que a mudança no tamanho do genoma apareceu em ambas as direções, enquanto os conteúdos de GC foram encontrados mais homogêneos no gênero Nicotiana.

The dynamics of genome size and GC contents evolution in genus Nicotiana / A dinâmica do tamanho do genoma e a evolução dos conteúdos de GC no gênero Nicotiana

Abstract Hybridization and Polyploidization are most common of the phenomenon observed in plants, especially in the genus Nicotiana leading to the duplication of genome. Although genomic changes associated with these events has been studied at various levels but the genome size and GC content variation is less understood because of absence of sufficient genomic data. In this study the flow cytometry technique was used to uncover the genome size and GC contents of 46 Nicotiana species and we compared the genomic changes associated with the hybridization events along evolutionary time scale. The genome size among Nicotiana species varied between 3.28 pg and 11.88 pg whereas GC contents varied between 37.22% and 51.25%. The tetraploid species in genus Nicotiana including section Polydiclae, Repandae, Nicotiana, Rustica and Sauveolentes revealed both up and downsizing in their genome sizes when compared to the sum of genomes of their ancestral species. The genome sizes of three homoploid hybrids were found near their ancestral species. Loss of large genome sequence was observed in the evolutionary more aged species (>10 Myr) as compared to the recently evolved one's (<0.2 Myr). The GC contents were found homogenous with a mean difference of 2.46% among the Nicotiana species. It is concluded that genome size change appeared in either direction whereas the GC contents were found more homogenous in genus Nicotiana.

Resumo A hibridização e a poliploidização são os fenômenos mais comuns observados em plantas, principalmente no gênero Nicotiana, levando à duplicação do genoma. Embora as mudanças genômicas associadas a esses eventos tenham sido estudadas em vários níveis, o tamanho do genoma e a variação do conteúdo de GC são menos compreendidos devido à ausência de dados genômicos suficientes. Neste estudo, a técnica de citometria de fluxo foi usada para descobrir o tamanho do genoma e o conteúdo de GC de 46 espécies de Nicotiana, e comparamos as mudanças genômicas associadas aos eventos de hibridização ao longo da escala de tempo evolutiva. O tamanho do genoma entre as espécies de Nicotiana variou entre 3,28 pg e 11,88 pg, enquanto os conteúdos de GC variaram entre 37,22% e 51,25%. As espécies tetraploides do gênero Nicotiana, incluindo as seções Polydiclae, Repandae, Nicotiana, Rustica e Sauveolentes, revelaram aumento e redução do tamanho do genoma quando comparados à soma dos genomas de suas espécies ancestrais. Os tamanhos do genoma de três híbridos homoploides foram encontrados perto de suas espécies ancestrais. A perda da grande sequência do genoma foi observada nas espécies evolutivas mais velhas (> 10 Myr) em comparação com as que evoluíram recentemente (< 0,2 Myr). Os teores de GC foram homogêneos com diferença média de 2,46% entre as espécies de Nicotiana. Conclui-se que a mudança no tamanho do genoma apareceu em ambas as direções, enquanto os conteúdos de GC foram encontrados mais homogêneos no gênero Nicotiana.

Extraction and bioactivity analysis of total protein of K326 tobacco leaves with high expression of Nicotiala alata defensin 1 gene / 中国生物制品学杂志

@#Objective To extract the total protein of K326 tobacco leaves with high expression of Nicotiana alata defensin 1(NaD1) gene and analyze its bioactivity.Methods Total proteins were extracted from Nicotiana alata flowers,wild type(WT) and K326 tobacco leaves(transgenic) with high expression of NaD1 gene,and determined for the concentrations by Bardford method,while for the antibacterial activity against fungi by filter paper method,and for the inhibition activity on cancer cells(HeLa cells) by CCK-8.Results The total protein concentrations of Nicotiana alata flowers,WT and transgenic K326 tobacco leaves were 11.25,10.33 and 10.14 mg/mL,respectively.The antibacterial activity of total protein from transgenic K326 tobacco leaves against Candida albicans was(85.68±3.08)%,which was 1.33 and 1.14 times that of total protein from WT K326 tobacco leaves and Nicotiana alata flowers,respectively(F=15 339,P <0.05);The antibacterial activity against Fusarium oxysporum was(148.48±2.47)%,which was 1.09 and 1.08 times that of total protein from WT K326tobacco leaves and Nicotiana alata flowers,respectively(F=4.927,P <0.05).The IC_(50) value of transgenic K326 tobacco leaf protein on HeLa cells was the smallest(6.11 mg/mL),and the inhibitory activity was 1.56 and 1.21 times that of total protein of WT K326 tobacco leaves and Nicotiana alata flowers,respectively(F=89.748,P <0.05).Conclusion The total protein of K326 tobacco with high expression of NaD1 gene has good antibacterial and anticancer bioactivities,which provides an experimental basis for producing antibacterial and anticancer biological agents with tobacco as bioreactor.

The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology

BACKGROUND: Designed mimetic molecules are attractive tools in biopharmaceuticals and synthetic biology. They require mass and functional production for the assessment of upcoming challenges in the near future. The DARPin family is considered a mimetic pharmaceutical peptide group with high affinity binding to specific targets. DARPin G3 is designed to bind to the HER2 (human epidermal growth factor receptor 2) tyrosine kinase receptor. Overexpression of HER2 is common in some cancers, including breast cancer, and can be used as a prognostic and predictive tool for cancer. The chloroplasts are cost-effective alternatives, equal to, and sometimes better than, bacterial, yeast, or mammalian expression systems. This research examined the possibility of the production of the first antibody mimetic, DARPin G3, in tobacco chloroplasts for HER2 imaging in oncology. RESULTS: The chloroplast specific DARPin G3 expression cassette was constructed and transformed into N. tabacum chloroplasts. PCR and Southern blot analysis confirmed integration of transgenes as well as chloroplastic and cellular homoplasmy. The Western blot analysis and ELISA confirmed the production of DARPin G3 at the commercial scale and high dose with the rate of 20.2% in leaf TSP and 33.7% in chloroplast TSP. The functional analysis by ELISA confirmed the binding of IMAC purified chloroplast-made DARPin G3 to the extracellular domain of the HER2 receptor with highly effective picomolar affinities. The carcinoma cellular studies by flow cytometry and immunofluorescence microscopy confirmed the correct functioning by the specific binding of the chloroplast-made DARPin G3 to the HER2 receptor on the surface of HER2-positive cancer cell lines. CONCLUSION: The efficient functional bioactive production of DARPin G3 in chloroplasts led us to introduce plant chloroplasts as the site of efficient production of the first antibody mimetic molecules. This report, as the first case of the cost-effective production of mimetic molecules, enables researchers in pharmaceuticals, synthetic biology, and bio-molecular engineering to develop tool boxes by producing new molecular substitutes for diverse purposes.

Intoxicación severa por Nicotiana glauca (Palán Palán) con miocardio atontado / Severe poisoning by Nicotiana glauca (Palán Palán) with stunned myocardium

Resumen Nicotiana glauca también llamada Palán Palán, es un arbusto con hojas verdes azuladas y despulidas y una flor amarilla tubular pendulante que presenta alcaloides piridínicos, como nicotina, nornicotina, anatabina y anabastina (análogo estructural de la Nicotina). Se presenta el caso clínico de una paciente de 50 años con cuadro agudo de debilidad muscular generalizada que evoluciona con paro respiratorio, tras la ingesta accidental de una cantidad desconocida de hojas de Nicotiana glauca, cultivadas en una huerta hogareña mediante técnica de hidroponía y confundidas por su conviviente con espinaca. Presentó aumento de lactato y Troponina Ultra Sensible e Hipoquinesia Global de Ventrículo Izquierdo en el ecocardiograma, compatible con Aton tamiento Miocárdico (AM), que evolucionó favorablemente. Si bien hay pocos reportes, se han informado muertes de animales y humanos, tras la ingesta accidental de Nicotiana glauca. El inicio del cuadro es rápido, con patrón bifásico, con vómitos y estímulo simpático, seguido por bloqueo ganglionar y neuromuscular, pudiendo presentar paro respiratorio, shock, convulsiones y coma. El AM es una disfunción miocárdica prolongada con retorno gradual de la actividad contráctil, posterior a un episodio breve de isquemia grave, puede ser asintomático, pudiendo presentar alteraciones en el electrocardiograma, enzimas cardíacas o ecocardiograma. Generalmente presenta pronóstico favorable, pudiendo presentar insuficiencia cardíaca ante patologías concurrentes o aumento de requerimientos de oxígeno.

Abstract Nicotiana glauca is a shrub with bluish green leaves and a pendulous tubular yellow flower. It has pyridine alkaloids, such as nicotine, nornicotine, anatabine and anabastine (structural analog of Nicotine). We present the case of a 50 years old pa- tient with acute generalized muscle weakness that evolves to respiratory arrest, after accidentally ingesting an unknown quantity of Nicotiana glauca leaves, grown in a home vegetable garden, using a hydroponic technique and confused by her cohabiting with spinach. She presented increased lactate and Ultra Sensitive Troponin and Left Ventricular Global Hypokinesia in the echo- cardiogram, compatible with Myocardial Stunned, that it evolved favorably. There are few reports, animal and human deaths have been reported following accidental ingestion of Nicotiana glauca. The onset of the symptoms is early, with a biphasic pattern, with vomiting and sympathetic stimulation, followed by ganglionic and neuromuscular blockage and may present respiratory arrest, shock, seizures and coma. Myocardial Stunned is a prolonged myocardial dysfunction with gradual return of contractile activity after a brief episode of severe ischemia, it can be asymptomatic, and it can present alterations in the electrocardiogram, cardiac enzymes or echocardiogram. Generally presents a benign prognosis, being able to present heart failure with concurrent patholo- gies or increased requirements.

Overexpression of NtAGPase small subunit gene increases leaf starch content and tobacco biomass / 生物工程学报

Production of biofuels such as ethanol from non-grain crops may contribute to alleviating the global energy crisis and reducing the potential threat to food security. Tobacco (Nicotiana tabacum) is a commercial crop with high biomass yield. Breeding of starch-rich tobacco plants may provide alternative raw materials for the production of fuel ethanol. We cloned the small subunit gene NtSSU of ADP-glucose pyrophosphorylase (NtAGPase), which controls starch biosynthesis in tobacco, and constructed a plant expression vector pCAMBIA1303-NtSSU. The NtSSU gene was overexpressed in tobacco upon Agrobacterium-mediated leaf disc transformation. Phenotypic analysis showed that overexpression of NtSSU gene promoted the accumulation of starch in tobacco leaves, and the content of starch in tobacco leaves increased from 17.5% to 41.7%. The growth rate and biomass yield of the transgenic tobacco with NtSSU gene were also significantly increased. The results revealed that overexpression of NtSSU gene could effectively redirect more photosynthesis carbon flux into starch biosynthesis pathway, which led to an increased biomass yield but did not generate negative effects on other agronomic traits. Therefore, NtSSU gene can be used as an excellent target gene in plant breeding to enrich starch accumulation in vegetative organs to develop new germplasm dedicated to fuel ethanol production.

Leaf area estimation of Burley tobacco / Estimativa da área foliar de tabaco do tipo Burley

ABSTRACT: Leaf area is an important growth variable in agricultural crops and the leaf is the main variable of interest in the tobacco industry. So, the aim of this scientific research was to estimate the Burley tobacco leaf area by linear dimensions of the leaves and to determine which mathematical model is more adequate for this purpose. Two experiments were carried out with Burley tobacco, cultivar DBH 2252, in 2016/2017 and 2018/2019 agricultural years, respectively, in the municipalities of Itaqui and Vanini - RS - Brazil. In 600 leaves were measured length (L), width (W), length×width product (LW), length/width ratio (L/W) and determined the real leaf area (LA). Four hundred and fifty leaves were separated to generate models of the leaf area as a function of linear dimension and the other 150 leaves were used for model's validation. The power model LA = 0.5037LW1.04435 (R² = 0.9960) is the most adequate for Burley tobacco 'DBH 2252' leaf area estimation. Alternatively, the models LA=2.0369W1.8619 (R²=0.9796) and LA=0.1222L2.2771 (R²=0.9738) based on width and length, respectively, can be used when only one leaf dimension is measured.

RESUMO: A área foliar é uma importante variável de crescimento em culturas agrícolas, sendo a folha a principal variável de interesse na indústria do tabaco. Assim, o objetivo deste estudo científico foi determinar a área foliar de tabaco tipo Burley por meio de dimensões lineares da folha e determinar qual modelo matemático é mais adequado para essa finalidade. Dois experimentos foram conduzidos com tabaco tipo Burley, cultivar DBH 2252, nos anos agrícolas de 2016/2017 e 2018/2019, respectivamente, nos municípios de Itaqui e Vanini, RS, Brasil. Em 600 folhas foram medidos o comprimento (L), a largura (W), o produto comprimento×largura (LW), a razão comprimento/largura (L/W) e determinada a área foliar real (LA). Foram separadas 450 folhas para a geração de modelos de estimativa de área foliar em função da dimensão linear e 150 folhas foram utilizadas para a validação dos modelos. O modelo LA = 0,5037LW1,04435 (R² = 0,9960) é adequado para a estimação da área foliar de tabaco Burley cultivar DBH 2252. Alternativamente, os modelos LA=2,0369W1,8619 (R²=0,9796) e LA=0,1222L2,2771 (R²=0,9738) baseados na largura e comprimento, respectivamente, podem ser utilizados quando apenas uma dimensão da folha é medida.

Identification, characterization and expression analysis of sucrose transporters in the model plant Nicotiana tabacum cv.petit havana

Aim: Sucrose transporters (SUTs) are transmembrane proteins involved in transport of sucrose along the phloem pathway of plants. Nicotiana tabacum, a well-known model plant has been reported to have three sucrose transporters (NtSUT1, NtSUT3 and NtSUT4), till date. The present study was undertaken to identify new SUTs from its genome and characterize all the NtSUT genes of Nicotiana tabacumMethodology: In the present study, new putative sucrose transporter genes were identified through extensive searches of the draft genome and transcriptome of tobacco using BLAST. In-silico characterization of the SUT gene family (NtSUT1, NtSUT2, NtSUT3, NtSUT4 and NtSUT5) of Nicotiana tabacum was done using bioinformatics tools. Spatial expression analysis was done for NtSUT genes in five different tissues of Nicotiana tabacum cv.petit havana using semi-quantitative RT-PCR. Results: Two new putative sucrose transporters NtSUT2 and NtSUT5 were identified from the publicly available tobacco transcriptome data and characterized using in-silico tools. Multiple sequence alignment of deduced amino acids of all the five SUTs revealed the presence of highly conserved domains and signature histidine residue in the sugar binding motif. Phylogenetic analysis grouped NtSUT1 and 3 into group 2, NtSUT4 and 5 into group 4 and NtSUT2 into group 3. NtSUT1 showed higher expression in leaves and flower while NtSUT2, 3 and 4 were abundant in root and flower. NtSUT5 showed a lower expression in all the tissues. Interpretation: Based on the sequence information and structural similarities, it is clear that the two newly identified putative SUT genes (NtSUT2 and NtSUT5) belonged to the SUT family of tobacco. This comprehensive study provides a consolidated data on characteristic features of SUT family proteins of tobacco

Tobacco waste hydrolysate of stem and root of the tobacco plant for biostimulation in rice and corn seed germination / Hidrolisado de resíduo de caule e raiz da planta tabaco para bioestimulação na germinação de sementes de arroz e milho

ABSTRACT: Stimulation of seed germination may be due to acceleration of germination as well as due to seedling growth-promotion during early development. Plant hydrolysate can be applied as a stimulant. Thus, we aimed to verify the influence of the hydrolysates, obtained by alkaline or acid treatment, from tobacco (Nicotiana tabacum L.) crop residues (root and stem) on the seed germination process. Seed germination was studied with Oryza sativa (rice) and Zea mays (corn). Sixteen germination experiments of 50 seeds each were undertaken, with 4 replicates, soaked with hydrolysates diluted at 20 and 80%, in 2 and 3 mL of hydration volumes for 48 h. Germinated seeds were counted, at which point radicular protrusions were observed. Rootlets and aerial parts were collected, dried and weighed. The hydrolysates presented seedling nutrition potential to the corn, with ~50% more mass compared to the results with water at the same conditions, and the germination acceleration was not significant. For the tested rice seeds, the results were reversed, and the germination acceleration was significant with rates up to 94% after 48 h of incubation. Better results of germination were obtained with hydrolysate from acid treatment, and root or stem tobacco can be used for this purpose.

RESUMO: A estimulação da germinação das sementes pode ser devida à aceleração da germinação, bem como à melhoria do crescimento das mudas durante o desenvolvimento inicial. O hidrolisado de plantas pode ser aplicado como estimulante. Assim, objetivou-se verificar a influência dos hidrolisados, obtidos por tratamento alcalino ou ácido, dos resíduos da cultura do tabaco (Nicotiana tabacum L.) (raiz e caule) no processo de germinação das sementes. A germinação das sementes foi estudada com Oryza sativa (arroz) e Zea mays (milho). Foram realizados dezesseis experimentos de germinação de 50 sementes cada, com 4 repetições, hidratadas em hidrolisados diluídos a 20 e 80%, em 2 e 3 mL de volumes de hidratação por 48 h. As sementes germinadas foram contadas, momento em que foram observadas protrusões radiculares. Radículas e partes aéreas foram coletadas, secas e pesadas. Os hidrolisados apresentaram potencial nutricional de plântulas para o milho, com ~ 50% a mais de massa quando comparados aos resultados com água nas mesmas condições, e a aceleração da germinação não foi significativa. Para as sementes de arroz testadas, os resultados foram contrários e a aceleração da germinação foi significativa com taxas de até 94% após 48 h de incubação. Melhores resultados de germinação foram obtidos com hidrolisado a partir do tratamento ácido, e a raiz ou caule de tabaco pode ser utilizado para esse fim.

Polyphenols and triterpenes in leaves and extracts from three Nicotiana species

Genus Nicotiana (Solanaceae) includes 76 species, among which only Nicotiana tabacum L. (common tobacco) isrecognized as economically important. Nicotiana species synthesize various secondary metabolites with biological orecological activities, but many of them still have not been sufficiently studied. The objective of current study was todetermine the content of selected groups of metabolites (e.g., phenolic acids, flavonoids, and triterpenes) in the leavesand extracts of three Nicotiana species (Nicotiana alata Link and Otto, Nicotiana rustica L., and N. tabacum L.),grown side by side. The results from the high-performance liquid chromatography identification of polyphenols andtriterpenes in leaves and extracts showed considerable variations between the species. All of them, however, werecharacterized as plant materials containing various specialized and bioactive metabolites, which substantiate theirconsideration as a valuable source for obtaining different phytoproducts. In terms of the studied groups of bioactivemetabolites, the results from the study suggest that 50% and 70% ethanol are the most suitable solvent concentrationsfor the extraction of N. alata, N. rustica, and N. tabacum leaves. The high content of polyphenols and triterpenesin the extracts provides grounds for their use in various cosmetic and pharmaceutical products, which could be thesubject of future research.

Development of KASP marker for cytoplasmic male sterility in Nicotiana tabacum and utilization in trait introgression

Cytoplasmic male sterility (CMS) is an important trait for large-scale hybrid seed production which avoids manual emasculation and undesired horizontal spread of pollen.Rearrangements in mitochondrial genome in terms of deletions and insertions are frequent causes leading to CMS. Mitochondrial ATP synthase is a multisubunit molecular machine which is involved in synthesis of ATP. In this study, three mutations in ATPase subunit 6 were identified and their cosegregation with male sterility was established using tobacco male sterile hybrids and Nicotiana suvaolensis. A breeder friendly Kompetitive allele specific polymerase chain reaction (KASP) SNP marker was developed for high throughput and quick genotyping. Introgression of this trait into selected germplasm lines (n = 9) was achieved based on foreground for CMS and background selection for recurrent parent using KASP marker and 50K custom tobacco SNP array, respectively. Analysis of genotyping data from SNP array revealed the presence of 88–99% of recurrent parent genome in BC3F1 plants. The selected BC3F1 plants exhibit CMS and are indistinguishable from the fertile recurrent parent (germplasm) in terms of plant morphology.

Histopathological Evaluation of the Efficacy for Plant-produced E2 Protein Vaccine against Classical Swine Fever Virus (CSFV) in Piglets

Classical swine fever (CSF), previously known as hog cholera, remains one of the most important swine-related contagious diseases worldwide. In order to eradicate classical swine fever virus (CSFV), it is commonly used in LOM-850 strain as a live attenuated CSF vaccine. However, there are symptoms of vaccination, such as the depression of feed intake, and difficulty of differentiation between infected and vaccinated hosts is impossible based on the antibodies induced. Nicotiana benthamiana were considered as an alternative to the production of recombinant vaccines on account of higher yields and levels of soluble protein than other models and crops in protein recombinant products. This study was conducted to evaluate histopathological validation of the plant-produced E2 fusion protein (ppE2) in piglets. The piglets were challenged by an injection of YC11WB strain in 7 days, 11 days and 14 days after one shot of the vaccination. The histopathological examination indicated that ppE2 can protect against lethal CSFV challenge at least 11 days of vaccination in piglets. These data suggest that the ppE2 can be an effective vaccine against CSFV in piglets.

Transient expression of hemagglutinin antigen from canine influenza virus H3N2 in Nicotiana benthamiana and Lactuca sativa

PURPOSE: Canine influenza virus (CIV), H3N2, carries potentiality for zoonotic transmission and genetic assortment which raises a concern on possible epidemics, and human threats in future. To manage possible threats, the development of rapid and effective methods of CIV vaccine production is required. The plant provides economical, safe, and robust production platform. We investigated whether hemagglutinin (HA) antigen from Korea-originated CIV could be produced in Nicotiana benthamiana and lettuce, Lactuca sativa by a DNA viral vector system. MATERIALS AND METHODS: We used DNA sequences of the HA gene from Korean CIV strain influenza A/canine/Korea/S3001/2015 (H3N2) for cloning into a geminiviral expression vectors to express recombinant HA (rHA) antigen in the plant. Agrobacterium-mediated infiltration was performed to introduce HA-carrying vector into host plants cells. Laboratory-grown N. benthamiana, and grocery-purchased or hydroponically-grown lettuce plant leaves were used as host plants. RESULTS: CIV rHA antigen was successfully expressed in host plant species both N. benthamiana and L. sativa by geminiviral vector. Both complex-glycosylated and basal-glycosylated form of rHA were produced in lettuce, depending on presence of endoplasmic reticulum (ER) retention signal. In terms of rHA expression level, canine HA (H3N2) showed preference to the native signal peptide than ER retention signal peptide in the tested geminiviral vector system. CONCLUSION: Grocery-purchased lettuce leaves could serve as an instant host system for the transient expression of influenza antigen at the time of emergency. The geminiviral vector was able to induce expression of complex-glycosylated and basal-glycosylated rHA in lettuce and tobacco.

Chemical Constituents of the Essential Oil Extracted from Nicotiana tabacum Leaves

Gas Chromatography-Mass Spectrometric (GC-MS) was used to investigate the chemical constituents of the essential oil from fresh leaves of Nicotiana tabacum L. The result revealed thirty six (36) compounds representing 100% of the essential oil characterized from the leaves of Nicotiana tabacum L. The major constituents are 3, 7, 11, 15-Tetramethyl-2-hexadecen-1-ol 16.37%, P-xylene 12.37%, Cyclohexane 8.43%, Farnesol isomer a 3.08%, and 1-Naphthalenepropanol 3.08%.

A new flavone from leaves of Nicotiana tabacum and its antibacterial activity / 中草药

Objective To find the new biological compounds, the research of the chemical constituents in the leaves of Nicotiana tabacum had been carried out. Methods The chemical constituents in the leaves of N.tabacum were isolated by silica gel, Sephadex LH-20 column chromatographies, and RP-HPLC methods. The structures of isolations were detected by using various spectroscopic techniques, including extensive 1D and 2D NMR techniques. Results Two C-alkylated flavones were isolated from 95% ethanol extract of plant, which identified as 2’-hydroxy-7-(3-hydroxypropyl)-8-methoxy-flavone (1) and tabaflavone A (2). Compounds 1 and 2 were evaluated for their anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) activity. Conclusion Compound 2 is isolated from the leaves of N.tabacum for the first time. Compound 1 is a new compound named orientalflavone A. Compounds 1 and 2 also show good anti-MRSA activity with MIC90 value of (38 ± 4) and (33 ± 3) μg/mL, respectively.

Ethnobotanical inventory of medicinal plants used in the Qampaya District, Bolivia / Inventario etnobotánico de plantas medicinales usadas en el Distrito Qampaya, Bolivia

Herbal medicine therapy is traditionally practiced by indigenous healers in Bolivia for hundreds years. Due to the great geographical and ecological diversity, there are thousands of native plants, which are utilized against diverse types of diseases. Nowadays, there is a worldwide problem connected with possible loss of ethnobotany knowledge because of the lack of the interest of young people. In present study, survey focused on medicinal plants used by rural people in Qampaya District, Potosí Department, Bolivia has been done. The data were collected from 60 respondents by semi-structured interviews. The results showed that 60 plant species belonging to 30 families are known as curative plants in this area. The mostly named families were Asteraceae (14 species) followed by Lamiaceae (7 species) and Brassicaceae (4 species). Predominant health problems treated by these plants are urological problems and gastro-intestinal disorders. The most frequently used plant parts were leaves and the preparation is mostly done as infusion. Even though the knowledge of using medical plants plays important role in life of Bolivian rural people, which use plants as medicines against various types of diseases, this study showed that 25 percent of respondents didn’t know any medicinal plants. On the other hand 40 percent of asked people have known 6 or more medicinal plant species.

La terapia de la medicina herbolaria es practicada, tradicionalmente, por los curanderos indígenas en Bolivia, desde hace cientos de años. Gracias a la gran diversidad geográfica y ecológica, hay miles de plantas nativas, que se utilizan para el tratamiento de diferentes tipos de enfermedades. Hoy en día, hay un problema mundial relacionado con la posible pérdida del conocimiento etnobotánico, esto debido a la falta de interés de las nuevas generaciones. El presente estudio descriptivo, mediante una encuesta, fue enfocado a las plantas medicinales utilizadas por la población rural del Distrito Qampaya, Departamento de Potosí, Bolivia. Los datos se obtuvieron de 60 informantes mediante una encuesta semiestructurada. Los resultados mostraron que 60 especies de plantas medicinales. pertenecientes a 30 familias botánicas, son conocidas en el área de intervención. Las familias botánicas, más importantes de uso medicinal son Asteraceae (14 especies), seguido de Lamiaceae (7 especies) y Brassicaceae (4 especies). Los problemas predominantes de salud, tratados con estas especies, son los urológicos y trastornos gastrointestinales. La parte de la planta más utilizada son las hojas y la forma más común de uso es en infusión. A pesar de que el conocimiento sobre la utilización de las plantas medicinales, juega un papel importante en la vida de la población rural de Bolivia, quienes utilizan las plantas medicinales para el tratamiento de diversos tipos de enfermedades, este estudio mostró que el 25 por ciento de los encuestados no tienen conocimiento sobre la utilidad medicinal de ninguna especie vegetal. Por otro lado, el 40 por ciento de los informantes conocen 6 o más especies de plantas medicinales.

A new diterpene from leaves of Nicotiana tabacum / 中草药

Objective: To study the cembranoid diterpenes from the leaves of Nicotiana tabacum. Methods: The cembranoid diterpenes were isolated and purified by silica gel and preparative HPLC, and their structures were elucidated on the basis of their physicochemistry properties and spectra data. Results: Five cembranoid diterpenes were obtained from the ethanol extract of the leaves of N. tabacum and identified as (1R, 3E, 7E, 10S, 11E)-3,7,11-cembratriene-10, 15-diols (1), (1S, 2E, 4S, 6R, 7E, 10E, 12S)-2, 7, 10- cembratriene-4, 6, 11-triol (2), (1S, 2E, 4R, 6R, 7E, 10E, 12S)-2, 7, 10-cembratriene-4, 6, 11-triol (3), (1S, 2E, 4S, 6R, 7E)-4, 6, 11-trihydroxy- 1-isopropyl-4, 8-dimethylpentadeca-2, 7-dien-12-one (4), and (1S, 2E, 4R, 6R, 7E, 11S, 12S)-11, 12-epoxy-2, 7-cembradiene-4, 6-diol (5). Conclusion: Compound 1 is a new cembranoid diterpene and named as nicotiaditerpene B, and compound 4 is a new natural product.

Detection of Serum Antibodies to Hepatitis E Virus Based on HEV Genotype 3 ORF2 Capsid Protein Expressed in Nicotiana benthamiana

BACKGROUND: Hepatitis E virus (HEV) causes epidemics in developing countries and is primarily transmitted through the fecal-oral route. There have been recent reports on the zoonotic spread of the virus, and several animal species, primarily pigs, have been recognized as reservoirs of HEV. Because of its possible spread, there is an urgent need of a method for the cost-effective production of HEV proteins that can be used as diagnostic antigens for the serological detection of anti-HEV antibodies. METHODS: The HEV open reading frame (ORF)2 protein was purified from plant tissue by using immobilized metal-anion chromatography (IMAC). The recombinant protein was used to develop an in-house ELISA for testing anti-HEV antibodies in both human and swine sera. Thirty-six serum samples collected from patients with serologically proven HEV infection with commercial kits were tested for anti-HEV IgG antibodies by using the plant-expressed protein. Forty-five serum samples collected from apparently healthy pigs in Bulgarian farms were also tested. RESULTS: We confirmed the transient expression and purification of a truncated version of the HEV genotype 3 capsid protein in Nicotiana benthamiana and its usefulness as a diagnostic antigen. ELISA showed the presence of anti-HEV IgG antibodies in 29 of the 36 human samples. The in-house ELISA showed anti-HEV IgG antibodies in 34 of the 45 pigs. CONCLUSIONS: We describe a method for the production of HEV ORF2 protein in N. benthamiana and the usefulness of this protein for the serological detection of anti-HEV antibodies in both humans and swine.

Qualidade de sementes de tabaco durante o processo de pelotização e armazenamento / Quality of tobacco seeds during the pelletizing process and storage

RESUMO: O processo de pelotização pode afetar a qualidade das sementes por ocasião da semeadura, por isso, para avaliar o efeito desse processo na qualidade das sementes, foram utilizadas sementes de duas cultivares de tabaco 'CSC 467' e 'CSC 07', coletadas em diferentes etapas do processo de beneficiamento e pelotização: 1) sementes nuas não beneficiadas; 2) sementes nuas beneficiadas; 3) sementes pelotizadas; 4) sementes pelotizadas e coloridas. Para avaliação da qualidade das sementes nessas diferentes etapas, foram realizados os seguintes testes: germinação, primeira contagem de germinação, índice de velocidade de germinação, teste de emergência e índice de velocidade de emergência. Posteriormente, as sementes foram armazenadas por seis e doze meses e sua qualidade avaliada pelos mesmos testes e determinações. O processo de pelotização avaliado não influencia na germinação final de sementes de tabaco, mas atrasa o processo germinativo. As sementes de tabaco das cultivares 'CSC 467' e 'CSC 07' mantêm sua qualidade durante os 12 meses de armazenamento em câmara fria, com temperatura de 10°C e umidade relativa de 50%.

ABSTRACT: The pelletizing process may affect the quality of seeds during sowing. In order to evaluate the effect of this process on seed quality it was used seeds from three tobacco cultivars, 'CSC 467' and 'CSC 07' collected in different processing and pelletizing process stages: 1) non processed nude seeds; 2) processed nude seeds; 3) pelletized seeds; 4) pelletized and colored seeds. For the evaluation of seed quality in these different stages it was performed the following tests: Germination, First Count of Germination, Germination Speed Index, Initial and Final Stand (Emergence) and Emergence Speed Index. Posteriorly, seeds were stored for six and twelve months and their quality was evaluated by the same tests and determinations. The evaluated pelletizing process does not influence final germination of the tobacco seeds, but delays the germination process. Tobacco seeds from cultivars 'CSC 467' and 'CSC 07' maintain their quality during the 12 months of storage in cold chamber with temperature of 10oC and relative humidity of 50%.