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Id: biblio-1046711 Autor: Yang Chee, Marcus Jenn; Lycett, Grantley W; Chin, Chiew Foan. Título: Development of a direct transformation method by GFP screening and in vitro whole plant regeneration of Capsicum frutescens L Fonte: Electron. j. biotechnol;34:51-58, july. 2018. ilus, tab, graf. Idioma: en. Projeto: Malaysian Ministry of Science, Technology and Innovation through the eScience Fund. Resumo: Background: Capsicum is a genus of an important spice crop that belongs to the chili lineage. However, many Capsicum species (family Solanaceae) are known to be recalcitrant to genetic transformation and in vitro regeneration, thus hampering the effort in using Capsicum species for detailed biological investigation. In this study, we have developed an optimized protocol for the direct transformation of Capsicum frutescens L. cv. Hot Lava using a biolistic particle delivery system. In addition, a procedure for in vitro whole plant regeneration from the hypocotyl explants of C. frutescens was established. Results: In this study on the biolistic system, explant target distance, bombardment helium (He) pressure, and the size of the microcarrier were the key parameters to be investigated. The optimized parameters based on the screening of GFP expression were determined to have a target distance of 6 cm, helium pressure of 1350 psi, and gold particle (microcarrier) size of 1.6 µm. The greatest number of shoots was obtained from hypocotyls as explants using Murashige and Skoog medium supplemented with 5.0-mg/L 6-benzylaminopurine and 0.1-mg/L 1-naphthaleneacetic acid. On an average, five shoots per explant were formed, and of them, one shoot managed to form the root and developed into a whole plant. Conclusions: We obtained an optimized protocol for the biolistic transformation of chili and in vitro regeneration of chili plantlets. The establishment of the protocols will provide a platform for molecular breeding and biological studies of chili plants. Descritores: Capsicum/crescimento & desenvolvimento -Regeneração Transformação Genética Técnicas In Vitro Capsicum/genética Reação em Cadeia da Polimerase Biolística Proteínas de Fluorescência Verde Técnicas de Cultura de Tecidos Engenharia Metabólica Responsável: CL1.1 - Biblioteca Central

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Id: biblio-1177370 Autor: Arévalo Gallegos, Sigifredo; Varela Rodríguez, Hugo; Lugo Aguilar, Héctor; Siqueiros Cendón, Tania S; Iglesias Figueroa, Blanca F; Espinoza Sánchez, Edward A; Aguado Santacruz, Gerardo A; Rascón Cruz, Quintín. Título: Transient expression of a green fluorescent protein in tobacco and maize chloroplast Fonte: Electron. j. biotechnol;45:1-9, May 15, 2020. ilus. Idioma: en. Resumo: BACKGROUND: Maize is one of the most important crops worldwide and has been a target of nuclear-based transformation biotechnology to improve it and satisfy the food demand of the ever-growing global population. However, the maize plastid transformation has not been accomplished due to the recalcitrant condition of the crop. RESULTS: In this study, we constructed two different vectors with homologous recombination sequences from maize (Zea mays var. LPC13) and grass (Bouteloua gracilis var. ex Steud) (pZmcpGFP and pBgcpGFP, respectively). Both vectors were designed to integrate into rrn23S/rrn16S from an inverted repeat region in the chloroplast genome. Moreover, the vector had the mgfp5 gene driven by Prrn, a leader sequence of the atpB gene and a terminator sequence from the rbcL gene. Also, constructs have an hph gene as a selection marker gene driven by Prrn, a leader sequence from rbcL gene and a terminator sequence from the rbcL gene. Explants of maize, tobacco and Escherichia coli cells were transformed with both vectors to evaluate the transitory expression­an exhibition of green and red fluorescent light under epifluorescence microscopy. These results showed that both vectors were expressed; the reporter gene in all three organisms confirmed the capacity of the vectors to express genes in the cell compartments. CONCLUSIONS: This paper is the first report of transient expression of GFP in maize embryos and offers new information for genetically improving recalcitrant crops; it also opens new possibilities for the improvement in maize chloroplast transformation with these vectors. Descritores: Tabaco/metabolismo Cloroplastos/genética Cloroplastos/metabolismo Zea mays/genética Proteínas de Fluorescência Verde/metabolismo -Transformação Genética Biotecnologia Reação em Cadeia da Polimerase Plantas Geneticamente Modificadas Plastídeos/genética Proteínas de Fluorescência Verde/genética Escherichia coli Genoma de Cloroplastos Responsável: CL1.1 - Biblioteca Central

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Id: biblio-1132162 Autor: Singh, Ravi Shankar; Jha, Vikash Kumar; Chattopadhyay, Tirthartha; Kumar, Ujjwal; Fulzele, Devanand Pralhad; Singh, Prabhash Kumar. Título: First Report of Agrobacterium rhizogenes-induced Hairy Root Formation in Selaginella bryopteris: a Pteridophyte Recalcitrant to Genetic Transformation Fonte: Braz. arch. biol. technol;63:e20180679, 2020. tab, graf. Idioma: en. Projeto: Board of Research in Nuclear Sciences (BRNS), Department of Atomic Energy (DAE). Resumo: Abstract we report A. rhizogenes-induced hairy root formation in S. bryopteris, a medicinally and commercially important plant. A. rhizogenes strain LBA1334 co-cultivated with explants (root, rhizophore, stem portion near the root, and stem with intact fronds) for 24 and 48 h after transformation for induction of hairy roots. The induction of hairy root was observed after 6 days of infection in case of 48 h co-cultivation only. PCR with rolA and virC gene specific primers confirmed the induced hairy roots were due to Ri T-DNA integration and not due to contaminating A. rhizogenes. The root network as explants showed the maximum transformation efficiency. We tested different media like MS, SHFR (Stage Hog Fern Root) and KNOP's during transformation for hairy root induction. The SHFR based media showed good response in transformation as well as propagation. Further, transformation efficiency was enhanced by addition of TDZ (2 mg/L) and Bevistin (0.1%) in SHFR media. The present work would be helpful in hairy roots-based in vitro production of secondary metabolites and on aspect of functional genomics of S. bryopteris. Descritores: Transformação Genética/genética Reação em Cadeia da Polimerase Selaginellaceae/microbiologia Agrobacterium/genética -Genômica Responsável: BR1.1 - BIREME

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Id: biblio-1053457 Autor: Suwanseree, Valerie; Phansiri, Salak; Yapwattanaphun, Chinawat. Título: A comparison of callus induction in 4 Garcinia species Fonte: Electron. j. biotechnol;40:45-51, July. 2019. ilus, tab. Idioma: en. Resumo: Background: This research is intended to determine suitable types and concentrations of plant growth regulators (PGRs) to induce callus on stem and leaf sections of 4 species of the genus Garcinia, namely, Garcinia mangostana, Garcinia schomburgkiana, Garcinia cowa, and Garcinia celebica. The base medium was MS medium containing 30 g l -1 sucrose, 0.5 g l-1 polyvinylpyrrolidone (PVP), and 7 g l-1 agar, and for the different treatments, PGRs were added to the medium as follows: thidiazuron (TDZ) at concentrations of 0, 0.1, 0.5, 1, and 2 mg l-1; 6-(3- hydroxybenzylamino) purine (meta-topolin) at concentrations of 0, 0.5, 2.5, and 5 mg l-1; 4-amino-3,5,6- trichloro-2-pyridinecarboxylic acid (picloram) at concentrations of 0, 0.5, 2.5, and 5 mg l-1; and 2,4- dichlorophenoxyacetic acid (2,4-D) at concentrations of 0, 0.5, 1, 2, and 4 mg l-1. The occurrence of callus was observed after 4 weeks. Results: A maximum of 100% and 93% of G. mangostana leaf explants formed callus in the 0.5 mg l-1 and 1 mg l-1 TDZ treatments, respectively, while 100% of G. schomburgkiana stem explants formed callus in the 1 mg l-1 TDZ treatment and 89% of G. schomburgkiana leaf explants formed callus in the 0.5 mg l-1 picloram treatment. The highest callus induction rate for G. cowa was 62% in the 1 mg l-1 TDZ treatment and for G. celebica was 56% in the 0.5 mg l-1•mT-1 treatment. Conclusions: For all 4 species, the greatest amount of large nodular callus was observed in the TDZ treatments. White, friable callus was observed on most of the 2,4-D and picloram treatment groups. Most meta-topolin treatments resulted in minimal callus formation. Descritores: Reguladores de Crescimento de Plantas/metabolismo Garcinia/crescimento & desenvolvimento Compostos Fitoquímicos/metabolismo -Compostos de Fenilureia Tiadiazóis Fatores de Tempo Transformação Genética Clusiaceae/crescimento & desenvolvimento Garcinia/fisiologia Técnicas de Cultura de Tecidos Responsável: CL1.1 - Biblioteca Central

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Id: biblio-1087255 Autor: Arencibia, Ariel D; D'Afonseca, Vívian; Chakravarthi, Mohan; Castiglione, Stefano. Título: Learning from transgenics: advanced gene editing technologies should also bridge the gap with traditional genetic selection Fonte: Electron. j. biotechnol;41:22-29, sept. 2019. ilus. Idioma: en. Resumo: We highlight the importance of the mixed genetic approaches (classical and currents) to improve the social perception related to the GMOs acceptance. We pointed out that CRISPR/Cas9 events could carry DNA variability/rearrangements related to somaclonal variations or epigenetic changes that are independent from the editing per se. The transformation of single cells, followed by plant regeneration, is used to generate modified plants, transgenic or genome editing (CRISPR/Cas9). The incidence of undesirable somaclonal variations and/or epigenetic changes that might have occurred during in vitro multiplication and regeneration processes, must be carefully analyzed in replicates in field trials. One remarkable challenge is related to the time lapse that selects the modified elite genotypes, because these strategies may spend a variable amount of time before the results are commercialized, where in all the cases it should be take into account the genotype × environment interactions. Furthermore, this combination of techniques can create an encouraging bridge between the public opinion and the community of geneticists who are concerned with plant genetic improvement. In this context, either transgenesis or genomic editing strategies become complementary modern tools to facing the challenges of plant genetic improvement. Their applications will depend on case-by-case analysis, and when possible will necessary associate them to the schemes and bases of classic plant genetic improvement. Descritores: Plantas Geneticamente Modificadas Técnicas de Transferência de Genes Sistemas CRISPR-Cas Edição de Genes -Transformação Genética Mutagênese Metilação de DNA Melhoramento Genético Epigênese Genética Tipo de Publ: Revisão Responsável: CL1.1 - Biblioteca Central

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Id: lil-129321 Autor: Villa, Luisa. Título: Epidemiología molecular y evolución de los papiloma virus humanos / Human Papillomavirus molecular epidemiology and evolution Fonte: Acta cancerol;23(3):13-8, set. 1993. ilus, tab, mapas. Idioma: es. Resumo: Hemos amplificado, clonado y secuenciado por la reacción en cadena de la polimerasa, los segmentos genómicos de 118 papiloma virus humanos tipo 16 (PVH-16) aislados de 76 biopsias cervicales, 14 exstendidos cervicales, 3 biopsias vulvares, 2 biopsias de pene, 2 biopsias anales, 1 biopsia vaginal y dos líneas celulares. Los especímenes fueron obtenidos de pacientes en cuatro países- Singapur, Brasil, Tanzania y Alemania. La secuencia de un fragmento de 364-bp de la región de control larga (RLC) de virus reveló métodos de distancia matriz y el enfoque de series de transformación. Los árboles basados en la RLV fueron confirmados por otro sset basado en la región completa. E5. ambos sets tuvieron dos ramas. Casi todas las variantes de Tanzania fueron asignadas a la rama africana, y todas las alemanas y al mayoría de las variantes de Singapur lo fueron a la rama eurasiática. En contraste con la homogeneidad interna de las variantes de Singapur Alemania y Tanzania, las variantes de Brasil estuvieron claramente divididas en las dos ramas. Los datos sugieren que el PVH-16 evolucionó separadamente por un tiempo largo en Africa y Eurasia. Representantes de ambas ramas podrían haber sido transferidos a Brasil vía la inmigración colonial. Representantes de la rama africana fueron posiblemente llevados al Lejano Este a través de las viejas rutas marinas árabes e indonesias. Nuestro estudio da soportes a la idea de que el PVH-16 es un tipo bien definido, pues las variantes muestran una divergencia genómica máxima de alrededor de 5 por ciento. La pequeña divergencia en cada localización geográfica y la falta de divergencia marcada entre las variantes genómica de Tanzania y la Brasilera-Africana luego de doscientos años desde su posible introducción en el Nuevo Mundo, sugieren una tasa muy lenta de evolución viral. El árbol filogenético probablemente representa un mínimo de varias centurias de evolución, si no una de igual a la de la raza humana. Descritores: Papillomaviridae/genética Variação Genética Evolução Biológica -Papillomaviridae/isolamento & purificação Transformação Genética Limites: Humanos Responsável: PE1.1 - Oficina Universitária de Biblioteca

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Id: biblio-1022493 Autor: Liu, Dongmei; Zhu, Hanyu; Chen, Yue; Zheng, Liesheng; Chen, Liguo; Ma, Aimin. Título: Cloning and heterologous expression of a hydrophobin gene Ltr. hyd from the tiger milk mushroom Lentinus tuber-regium in yeast-like cells of Tremella fuciformis Fonte: Electron. j. biotechnol;32:6-12, Mar. 2018. tab, graf, ilus. Idioma: en. Projeto: National Natural Science Foundation of China (NSFC). Resumo: Background: Hydrophobins are small proteins secreted by filamentous fungi, which show a highly surface activity. Because of the signally self-assembling abilities and surface activities, hydrophobins were considered as candidates in many aspects, for example, stabilizing foams and emulsions in food products. Lentinus tuber-regium, known as tiger milk mushroom, is both an edible and medicinal sclerotium-producing mushroom. Up to now, the hydrophobins of L. tuber-regium have not been identified. Results: In this paper, a Class I hydrophobin gene, Ltr.hyd, was cloned from L. tuber-regium and expressed in the yeast-like cells of Tremella fuciformis mediated by Agrobacterium tumefaciens. The expression vector pGEH-GH was under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter. The integration of Ltr.hyd into the genome of T. fuciformis was confirmed by PCR, Southern blot, fluorescence observation and quantitative real-time PCR (qRT-PCR). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that recombinant hydrophobin rLtr.HYD with an expected molecular mass of 13 kDa was extracted. The yield of rLtr.HYD was 0.66 mg/g dry weight. The emulsifying activity of rLtr.HYD was better than the typical food emulsifiers sodium caseinate and Tween 20. Conclusions: We evaluated the emulsifying property of hydrophobin Ltr.HYD, which can be potentially used as a food emulsifier. Descritores: Basidiomycota/metabolismo Proteínas Fúngicas/genética Lentinula/genética Lentinula/metabolismo -Transformação Genética Basidiomycota/enzimologia Leveduras Proteínas Fúngicas/metabolismo Southern Blotting Clonagem Molecular Agrobacterium tumefaciens/metabolismo Análise de Sequência Emulsificantes Eletroforese em Gel de Poliacrilamida Reação em Cadeia da Polimerase em Tempo Real Gliceraldeído-3-Fosfato Desidrogenases/metabolismo Microscopia de Fluorescência Responsável: CL1.1 - Biblioteca Central

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Id: biblio-1021034 Autor: Mihálik, Daniel; Gubisová, Marcela; Kraic, Ján; Hudcovicová, Martina; Havrlentová, Michaela; Moravcíková, Jana; Glasa, Miroslav; Matusíková, Ildikó. Título: Introduction of a synthetic Thermococcus-derived α-amlyase gene into barley genome for increased enzyme thermostability in grains Fonte: Electron. j. biotechnol;30:1-5, nov. 2017. ilus, tab, graf. Idioma: en. Projeto: Slovak Research and Development Agency. Resumo: Background: The enzymes utilized in the process of beer production are generally sensitive to higher temperatures. About 60% of them are deactivated in drying the malt that limits the utilization of starting material in the fermentation process. Gene transfer from thermophilic bacteria is a promising tool for producing barley grains harboring thermotolerant enzymes. Results: Gene for α-amylase from hydrothermal Thermococcus, optimally active at 75­85°C and pH between 5.0 and 5.5, was adapted in silico to barley codon usage. The corresponding sequence was put under control of the endosperm-specific promoter 1Dx5 and after synthesis and cloning transferred into barley by biolistics. In addition to model cultivar Golden Promise we transformed three Slovak barley cultivars Pribina, Levan and Nitran, and transgenic plants were obtained. Expression of the ~50 kDa active recombinant enzyme in grains of cvs. Pribina and Nitran resulted in retaining up to 9.39% of enzyme activity upon heating to 75°C, which is more than 4 times higher compared to non-transgenic controls. In the model cv. Golden Promise the grain α-amylase activity upon heating was above 9% either, however, the effects of the introduced enzyme were less pronounced (only 1.22 fold difference compared with non-transgenic barley). Conclusions: Expression of the synthetic gene in barley enhanced the residual α-amylase activity in grains at high temperatures. Descritores: Sementes/enzimologia Hordeum/enzimologia Thermococcus/metabolismo alfa-Amilases/metabolismo -Sementes/genética Sementes/microbiologia Transformação Genética Hordeum/genética Hordeum/microbiologia Cerveja Estabilidade Enzimática Plantas Geneticamente Modificadas/enzimologia Clonagem Molecular Técnicas de Transferência de Genes alfa-Amilases/genética Fermentação Termotolerância Temperatura Alta Concentração de Íons de Hidrogênio Responsável: CL1.1 - Biblioteca Central

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Id: biblio-1017249 Autor: Li, Tao; Ding, Yatong; Zhang, Jun; Jiao, Guobao; Sun, Lipeng; Liu, Zhongmin; Qiu, Liyou. Título: Improving the expression of recombinant pullulanase by increasing mRNA stability in Escherichia coli Fonte: Electron. j. biotechnol;29:63-67, sept. 2017. ilus, tab, graf. Idioma: en. Projeto: National Science and Technology Program in Rural Areas during the 12th Five-Year Plan period. Resumo: Background: Pullulanase production in both wild-type strains and recombinantly engineered strains remains low. The Shine-Dalgarno (SD) sequence and stem-loop structure in the 5' or 3' untranslated region (UTR) are well-known determinants of mRNA stability. This study investigated the effect of mRNA stability on pullulanase heterologous expression. Results: We constructed four DNA fragments, pulA, SD-pulA, pulA-3t, and SD-pulA-3t, which were cloned into the expression vector pHT43 to generate four pullulanase expression plasmids. The DNA fragment pulA was the coding sequence (CDS) of pulA in Klebsiella variicola Z-13. SD-pulA was constructed by the addition of the 5' SD sequence at the 5' UTR of pulA. pulA-3t was constructed by the addition of a 3' stem-loop structure at the 3' UTR of pulA. SD-pulA-3t was constructed by the addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of pulA. The four vectors were transformed into Escherichia coli BL21(DE3). The pulA mRNA transcription of the transformant harboring pHT43-SD-pulA-3t was 338.6%, 34.9%, and 79.9% higher than that of the other three transformants, whereas the fermentation enzyme activities in culture broth and intracellularly were 107.0 and 584.1 times, 1.2 and 2.0 times, and 62.0 and 531.5 times the amount of the other three transformants (pulA, SD-pulA, and pulA-3 t), respectively. Conclusion: The addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of the pulA gene is an effective approach to increase pulA gene expression and fermentation enzyme activity. Descritores: Escherichia coli/enzimologia Escherichia coli/genética Glicosídeo Hidrolases/metabolismo -Transformação Genética Expressão Gênica Reação em Cadeia da Polimerase Via Transcriptase Reversa Estabilidade de RNA Fermentação Vetores Genéticos Glicosídeo Hidrolases/genética Responsável: CL1.1 - Biblioteca Central

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Id: biblio-1009000 Autor: Liu, Chunming; Yang, Bowen; Ming, Yuetong; Liu, Jianfeng; Cheng, Yunqing. Título: Construction of an RNAi vector for knockdown of GM-ACS genes in the cotyledonary nodes of soybean Fonte: Electron. j. biotechnol;26:40-45, Mar. 2017. ilus, graf. Idioma: en. Projeto: National Natural Science Foundation of China; . Special Foundation for Young Scientists of Jilin Province, China. Resumo: Background: Ethylene plays an important role in the regulation of floral organ development in soybean, and 1-aminocyclopropane-1-carboxylate synthase (ACS) is a rate-limiting enzyme for ethylene biosynthesis. However, whether ACS also regulates floral organ differentiation in soybean remains unknown. To address this, we constructed an RNAi vector to inhibit ACS expression in cotyledonary nodes. Linear DNA cassettes of RNAi-ACS obtained by PCR were used to transform soybean cotyledonary nodes. Results: In total, 131 of 139 transiently transformed plants acquired herbicide resistance and displayed GUS activities in the new buds. In comparison to untransformed seedling controls, a greater number of flower buds were differentiated at the cotyledonary node; GM-ACS1 mRNA expression levels and ethylene emission in the transformed buds were reduced. Conclusion: These results indicate that the cotyledonary node transient transformation system may be suitable for stable transformation and that the inhibition of ACS expression may be an effective strategy for promoting floral organ differentiation in soybean. Descritores: Soja/enzimologia Soja/genética Interferência de RNA Liases/metabolismo -Soja/crescimento & desenvolvimento Transformação Genética Expressão Gênica Diferenciação Celular Reação em Cadeia da Polimerase Regulação da Expressão Gênica de Plantas Etilenos/biossíntese Resistência a Herbicidas Vetores Genéticos Glucuronidase Responsável: CL1.1 - Biblioteca Central

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