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Red de coexpresión de 320 genes de Tectona grandis relacionados con procesos de estrés abiótico y xilogénesis
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Camel, Vladimir

; Galeano, Esteban

; Carrer, Helaine

TIP. Revista especializada en ciencias químico-biológicas

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dec 2017, Volume 20 N. 2 elocation 10.1016/j.recqb.2017.04.001

Abstract: En Es | Text: En Es | PDF: En | PDF: Es

Abstract Teak (Tectona grandis) is a timber tree of economic importance in tropical and subtropical forests. The aim of this work was to identify families of transcription factors (TFs) and enzyme-coding genes differentially expressed (DREs) in stem xylem and their regulation involved in abiotic stress response and xylogenesis in T. grandis. Therefore, the evolutionary distribution of 19 TFs of T. grandis was derived using a phylogenetic analysis. Besides, specific data mining procedures of databases and publications were performed in order to identify 320 Arabidopsis thaliana genes (orthologous to T. grandis) as experimental and predictive support. As results, we found transcription factors of the bZIP, MYB, NAC, ER, bhlh families, and enzyme-coding genes. Furthermore, interactome analysis in T. grandis showed a significant Pearson correlation for genes regulating metabolic pathways of phenylpropanoids and abiotic stress. Also, the coexpression network revealed nodes and edges between TgRAP1, TgMyB1, TgHSF1, TgMyB3, TgNAC1, TgTsiid1, TgLieTFs1, TgNuy3, TgRAP2 and TgNuy4 genes. Gene ontology analyses showed that 31 genes respond to abiotic stress, mainly TgHShT1, TgHSF1 and TgHSF2, as co-regulators. In addition, the TFs master regulator TgNAC1 was found to be involved in the co-regulation of other TFs.

Resumen Tectona grandis es un árbol maderable de importancia económica en bosques tropicales y subtropicales. Mediante este estudio, se identificaron familias de factores de transcripción (FTs) y genes codificantes para enzima, diferencialmente expresados en el xilema del tallo, implicados en la regulación de la respuesta a estrés abiótico y xilogénesis en T. grandis. Así, fue analizada la distribución evolutiva de 19 genes codificantes para FTs de T. grandis mediante análisis filogenéticos. También, fue utilizada la minería de bases de datos y publicaciones para identificar 320 genes de Arabidopsis thaliana (ortólogos a T. grandis) como soporte experimental y predictivo. Como resultados, se encontraron FTs de las familias bZIP, MYB, NAC, ER, bhlh, NuY y genes que codifican enzimas. Así mismo, se logró analizar el interactoma de T. grandis encontrando correlaciones de Pearson significativas para genes que regulan vías metabólicas de fenilpropanoides y estrés abiótico. Además, la red de coexpresión reveló nodos y aristas entre los genes TgRAP1, TgMyB1, TgHSF1, TgMyB3, TgNAC1, TgTsiid1, TgLieTFs1, TgNuy3, TgRAP2 y TgNuy4. En particular, los análisis de ontología génica mostraron 31 genes de respuesta a estrés abiótico, principalmente TgHShT1, TgHSF1 y TgHSF2 como correguladores. Además, se encontró que el regulador maestro TgNAC1, está involucrado en la corregulación de otros factores de transcripción.

https://doi.org/10.1016/j.recqb.2017.04.001 818 downloads

ANÁLISIS in silico Y EXPRESIÓN GÉNICA DEL FACTOR DE TRANSCRIPCIÓN TgNACO1 IMPLICADO EN XILOGÉNESIS Y ESTRÉS ABIÓTICO EN Tectona grandis.
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CAMEL PAUCAR, Vladimir

; GALEANO, Esteban

; CARRER, Helaine

Acta Biológica Colombiana

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dec 2017, Volume 22 N. 3 Pages 359 - 369

Abstract: Es En | Text: Es En | PDF: Es | PDF: En

RESUMEN El xilema secundario es el componente más abundante de la biomasa vegetal. Por tanto, conocer los genes que regulan su formación ayudaría a diseñar estrategias para el mejoramiento genético de la madera. Así, el objetivo de este trabajo fue realizar el análisis computacional de la estructura primaria y secundaria del factor de transcripción (FT) TgNACO1 de Tectona grandis, además de evaluar su historia evolutiva, dominios conservados y expresión génica en tejidos lignificados de árboles de 12 y 60 años. Para ello, se realizó una evaluación del potencial de interacción ion-electrón (PIIE), mediante el método del espectro de la información (MEI) utilizando la librería SFAPS de R-Project, seguido del modelamiento estructural utilizando el software MODELLER y visualizado mediante PyMol. Además, el análisis de alineamiento de secuencia múltiple y filogenia fue mediante el software Bioedit y MrBayes respectivamente. También se evaluó los niveles de síntesis del FT TgNACO1 mediante qRT-PCR. Como resultados, se evidenció que el FT mantiene una estructura (3-hoja antiparalela retorcida, que se compacta contra una a-hélice en la región N-terminal, teniendo así tres dominios a hélice y siete dominios (3 plegada. Asimismo, mediante el MEI se demostró que tiene alrededor de cinco funciones biológicas y mutaciones sobre los aminoácidos con mayor PIIE, lo que conlleva a evoluciones sobre las redes de regulación genética. Finalmente, el FT TgNACO1 podría presentar un papel fundamental en la organización y desarrollo de las partes que componen la albura, como las células radiales de la zona cambial, los vasos, fibras y los anillos de crecimiento.

ABSTRACT Secondary xylem is the most abundant component of plant biomass. Therefore, knowing the genes that regulate its formation would help to design strategies for wood genetic improvement. Thus, the objective of this work was to perform computational analysis of the primary and secondary structure of the TgNACO1 transcription factor (FT) of Tectona grandis, and to evaluate its evolutionary history, conserved domains and gene expression in lignified tissues of trees with 12 and 60 years old. For this, an ion-electron interaction potential (IEP) was evaluated using the information-spectrum method (IEM) using the R-Project and SFAPS library, followed by structural modeling using the MODELLER software and visualized by PyMol program. In addition, the analysis of multiple sequence alignment and phylogeny was performed using Bioedit and MrBayes software, respectively. We also evaluated the qRT-PCR levels of TgNACO1. As results, it was found that TgNACO1 maintains a twisted antiparallel 3-sheet structure, which is compacted against an a-helix in the N-terminal region, having three a-helix domains and seven folded ((-domains. Also, through the IEM, it was demonstrated that it has about five biological functions, and mutations on amino acids with higher IEP, which leads to evolutions on genetic regulation networks. Finally, the FT TgNACO1 could play an esential role in the organization and development of the parts that make up the sapwood, such as the radial cells of the cambial zone, the vessels, fibers and the growth rings.

https://doi.org/10.15446/abc.v22n3.62164 1404 downloads

Efficient and new method for Tectona grandis in vitro regeneration
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Tambarussi, Evandro V.

; Rogalski, Marcelo

; Galeano, Esteban

; Brondani, Gilvano Ebling

; Martin, Valentina de Fatima de

; Silva, Lucas Américo da

; Carrer, Helaine

Crop Breeding and Applied Biotechnology

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jun 2017, Volume 17 N. 2 Pages 124 - 132

Abstract: En | Text: En | PDF: En

Abstract The aim of this study was to develop a shoot regeneration protocol for Tectona grandis by examining the effects of pretreatments, regeneration culture medium, adventitious rooting, and acclimatization. The best organogenic potential was achieved when hypocotyls and cotyledonary segments from seedlings germinated on a medium containing TDZ, were cultured for four weeks on MS medium supplemented with BAP/GA3. With TDZ pretreatment during seed germination, up to 70% of the cotyledonary segments (0.5 TDZ pretreatment and 1.0 BAP + 0.5 GA3 during regeneration, mg L-1) and 60% of the hypocotyl explants (0.1 TDZ pretreatment and 1.0 BAP + 0.5 GA3 during regeneration, mg L-1) resulted in shoot regeneration. Finally, 65% of the regenerated shoots rooted, of which 60% were successfully acclimatized ex vitro. Our protocol increases regeneration efficiency for teak and it can serve as a platform for genetic transformation to improve this important tree species.

https://doi.org/10.1590/1984-70332017v17n2a19 2730 downloads

Biotecnologia na agricultura
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Carrer, Helaine

; Barbosa, André Luiz

; Ramiro, Daniel Alves

Estudos Avançados

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2010, Volume 24 N. 70 Pages 149 - 164

Abstract: Pt En | Text: Pt En | PDF: Pt | PDF: En

A expectativa de o crescimento populacional atingir 9 bilhões de habitantes em 2050 em adição às questões da sustentabilidade e do aquecimento global nos desafiam a aumentar a oferta de alimentos. Uma metodologia alternativa que contribua para a redução do impacto desse cenário envolve a biotecnologia, que, nas últimas décadas, trouxe marcantes oportunidades tecnológicas na agricultura, resultando em relevante desenvolvimento na obtenção de novas variedades de plantas, na melhoria da qualidade de diversos alimentos e atualmente também na bioenergia. As técnicas biotecnológicas envolvendo os marcadores moleculares, a genômica e a transformação genética estão transformando a agricultura e são discutidas neste artigo.

The expected population growth to reach 9 billion by 2050 in addition to issues of sustainability and global warming challenges us to increase the supply of food. An alternative approach to help reducing the impact of this scenario involves biotechnology which in recent decades has brought remarkable technological opportunities in the agriculture that resulted in relevant development in obtaining new plant varieties, improved quality of different foods, and now also in bioenergy. The biotechnology techniques involving molecular markers, genomics and genetic transformation are transforming agriculture and will be discussed in this article.

180498 downloads

Signaling pathways in a Citrus EST database
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Mehta, Angela

; Silva, Marilia Santos

; Guidetti-Gonzalez, Simone

; Carrer, Helaine

; Takita, Marco Aurélio

; Martins, Natália F.

Genetics and Molecular Biology

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2007, Volume 30 N. 3 Pages 734 - 751

Abstract: En | Text: En | PDF: En

Citrus spp. are economically important crops, which in Brazil are grown mainly in the State of São Paulo. Citrus cultures are attacked by several pathogens, causing severe yield losses. In order to better understand this culture, the Millenium Project (IAC Cordeirópolis) was launched in order to sequence Citrus ESTs (expressed sequence tags) from different tissues, including leaf, bark, fruit, root and flower. Plants were submitted to biotic and abiotic stresses and investigated under different development stages (adult vs. juvenile). Several cDNA libraries were constructed and the sequences obtained formed the Citrus ESTs database with almost 200,000 sequences. Searches were performed in the Citrus database to investigate the presence of different signaling pathway components. Several of the genes involved in the signaling of sugar, calcium, cytokinin, plant hormones, inositol phosphate, MAPKinase and COP9 were found in the citrus genome and are discussed in this paper. The results obtained may indicate that similar mechanisms described in other plants, such as Arabidopsis, occur in citrus. Further experimental studies must be conducted in order to understand the different signaling pathways present.

3437 downloads

Identification of protein kinase SNF1 in CitEST
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Guidetti-Gonzalez, Simone

; Ragassi, Carlos Francisco

; Carrer, Helaine

Genetics and Molecular Biology

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2007, Volume 30 N. 3 Pages 866 - 871

Abstract: En | Text: En | PDF: En

SnRKs (Sucrose non-fermenting-1 related kinases) is a family of protein kinases found in many crops, such as Arabidopsis, rice, sugarcane, tomato and several other plant species. This family of proteins is also present in other organisms like Saccharomyces cerevisiae (sucrose non-fermenting-1 - Snf1) and in mammals (AMP-activated protein kinases - AMPKs). There is evidence that SnRKs play an important role in plant responses to nutritional and environmental stresses and that SnRKs also play a major role in controlling key enzymes in the biosynthetic pathways of plants. In this work, we identified 18 contigs and two singletons encoding putative SnRKs in the CitEST database. All of them present highly conserved N-terminal catalytic domain, which is found in the SnRKs families of several plant species. Through comparison with known SnRKs, we were able to classify them into three subfamilies.

2008 downloads

Putative resistance genes in the CitEST database
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Guidetti-Gonzalez, Simone

; Carrer, Helaine

Genetics and Molecular Biology

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2007, Volume 30 N. 3 Pages 931 - 942

Abstract: En | Text: En | PDF: En

Disease resistance in plants is usually associated with the activation of a wide variety of defense responses to prevent pathogen replication and/or movement. The ability of the host plant to recognize the pathogen and to activate defense responses is regulated by direct or indirect interaction between the products of plant resistance (R) and pathogen avirulence (Avr) genes. Attempted infection of plants by avirulent pathogens elicits a battery of defenses often followed by the collapse of the challenged host cells. Localized host cell death may help to prevent the pathogen from spreading to uninfected tissues, known as hypersensitive response (HR). When either the plant or the pathogen lacks its cognate gene, activation of the plant’s defense responses fails to occur or is delayed and does not prevent pathogen colonization. In the CitEST database, we identified 1,300 reads related to R genes in Citrus which have been reported in other plant species. These reads were translated in silico, and alignments of their amino acid sequences revealed the presence of characteristic domains and motifs that are specific to R gene classes. The description of the reads identified suggests that they function as resistance genes in citrus.

2947 downloads

Genes associated with hypersensitive response (HR) in the citrus EST database (CitEST)
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Guidetti-Gonzalez, Simone

; Freitas-Astúa, Juliana

; Amaral, Alexandre Morais do

; Martins, Natália F.

; Mehta, Angela

; Silva, Marilia Santos

; Carrer, Helaine

Genetics and Molecular Biology

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2007, Volume 30 N. 3 Pages 943 - 956

Abstract: En | Text: En | PDF: En

Plants are continuously exposed to pathogen attack, but successful infection is rare because they protect themselves against pathogens using a wide range of response mechanisms. One of them is the hypersensitive response (HR), which is a form of cell death often associated with plant resistance to pathogen infection to prevent the spreadsebpg@cnpq.br sebpg@cnpq.br of the potential pathogen from infected to uninfected tissues. Cell death is activated by recognition of pathogen-derived molecules by the resistance (R) gene products, and is associated with the massive accumulation of reactive oxygen species (ROS), salicylic acid (SA), and other pro-death signals such as nitric oxide (NO). The analysis of the citrus EST (CitEST) database revealed the presence of putative genes likely to be involved in HR through their products, like metacaspases, lipoxygenases, phospholipases, pathogenesis-related proteins, glutathione transferases/peroxidases, enzymes involved in the phenylpropanoid pathway and in the formation and detoxification of ROS, as well as those involved in the formation and regulation of ion channels, SA and NO. By analysis of the EST database of Citrus, it was possible to identify several putative genes that code for key enzymes involved in HR triggering and also in plant defense against biotic and abiotic stress.

4889 downloads

Brazilian coffee genome project: an EST-based genomic resource
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Vieira, Luiz Gonzaga Esteves

; Andrade, Alan Carvalho

; Colombo, Carlos Augusto

; Moraes, Ana Heloneida de Araújo

; Metha, Ângela

; Oliveira, Angélica Carvalho de

; Labate, Carlos Alberto

; Marino, Celso Luis

; Monteiro-Vitorello, Claúdia de Barros

; Monte, Damares de Castro

; Giglioti, Éder

; Kimura, Edna Teruko

; Romano, Eduardo

; Kuramae, Eiko Eurya

; Lemos, Eliana Gertrudes Macedo

; Almeida, Elionor Rita Pereira de

; Jorge, Érika C.

; Albuquerque, Érika V. S.

; Silva, Felipe Rodrigues da

; Vinecky, Felipe

; Sawazaki, Haiko Enok

; Dorry, Hamza Fahmi A.

; Carrer, Helaine

; Abreu, Ilka Nacif

; Batista, João A. N.

; Teixeira, João Batista

; Kitajima, João Paulo

; Xavier, Karem Guimarães

; Lima, Liziane Maria de

; Camargo, Luis Eduardo Aranha de

; Pereira, Luiz Filipe Protasio

; Coutinho, Luiz Lehmann

; Lemos, Manoel Victor Franco

; Romano, Marcelo Ribeiro

; Machado, Marcos Antonio

; Costa, Marcos Mota do Carmo

; Sá, Maria Fátima Grossi de

; Goldman, Maria Helena S.

; Ferro, Maria Inês T.

; Tinoco, Maria Laine Penha

; Oliveira, Mariana C.

; Van Sluys, Marie-Anne

; Shimizu, Milton Massao

; Maluf, Mirian Perez

; Eira, Mirian Therezinha Souza da

; Guerreiro Filho, Oliveiro

; Arruda, Paulo

; Mazzafera, Paulo

; Mariani, Pilar Drummond Sampaio Correa

; Oliveira, Regina L.B.C. de

; Harakava, Ricardo

; Balbao, Silvia Filippi

; Tsai, Siu Mui

; Mauro, Sonia Marli Zingaretti di

; Santos, Suzana Neiva

; Siqueira, Walter José

; Costa, Gustavo Gilson Lacerda

; Formighieri, Eduardo Fernandes

; Carazzolle, Marcelo Falsarella

; Pereira, Gonçalo Amarante Guimarães

Brazilian Journal of Plant Physiology

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mar 2006, Volume 18 N. 1 Pages 95 - 108

Abstract: Pt En | Text: Pt En | PDF: Pt | PDF: En

O café é um dos principais produtos agrícolas, sendo considerado o segundo item em importância do comércio internacional de "commodities". O gênero Coffea pertence à família Rubiaceae que também inclui outras plantas importantes. Este gênero contém aproximadamente 100 espécies, mas a produção comercial é baseada somente em duas espécies, Coffea arabica e Coffea canephora, que representam aproximadamente 70 % e 30 % do mercado total de café, respectivamente. O Projeto Genoma Café Brasileiro foi desenvolvido com o objetivo de disponibilizar os modernos recursos da genômica à comunidade científica e aos diferentes segmentos da cadeia produtiva do café. Para isso, foram seqüenciados 214.964 clones escolhidos aleatoriamente de 37 bibliotecas de cDNA de C. arabica, C. canephora e C. racemosa representando estádios específicos do desenvolvimento de células e de tecidos do cafeeiro, resultando em 130.792, 12.381 e 10.566 seqüências de cada espécie, respectivamente, após processo de trimagem. Os ESTs foram agrupados em 17.982 contigs e em 32.155 singletons. A comparação destas seqüências pelo programa BLAST revelou que 22 % não tiveram nenhuma similaridade significativa às seqüências no banco de dados do National Center for Biotechnology Information (de função conhecida ou desconhecida). A base de dados de ESTs do cafeeiro resultou na identificação de cerca de 33.000 unigenes diferentes. Os resultados de anotação das seqüências foram armazenados em base de dados "online" em <A HREF="http://www.lge.ibi.unicamp.br/cafe">http://www.lge.ibi.unicamp.br/cafe</A>. Os recursos desenvolvidos por este projeto disponibilizam ferramentas genéticas e genômicas que podem ser decisivas para a sustentabilidade, a competitividade e a futura viabilidade da agroindústria cafeeira nos mercados interno e externo.

Coffee is one of the most valuable agricultural commodities and ranks second on international trade exchanges. The genus Coffea belongs to the Rubiaceae family which includes other important plants. The genus contains about 100 species but commercial production is based only on two species, Coffea arabica and Coffea canephora that represent about 70 % and 30 % of the total coffee market, respectively. The Brazilian Coffee Genome Project was designed with the objective of making modern genomics resources available to the coffee scientific community, working on different aspects of the coffee production chain. We have single-pass sequenced a total of 214,964 randomly picked clones from 37 cDNA libraries of C. arabica, C. canephora and C. racemosa, representing specific stages of cells and plant development that after trimming resulted in 130,792, 12,381 and 10,566 sequences for each species, respectively. The ESTs clustered into 17,982 clusters and 32,155 singletons. Blast analysis of these sequences revealed that 22 % had no significant matches to sequences in the National Center for Biotechnology Information database (of known or unknown function). The generated coffee EST database resulted in the identification of close to 33,000 different unigenes. Annotated sequencing results have been stored in an online database at <A HREF="http://www.lge.ibi.unicamp.br/cafe">http://www.lge.ibi.unicamp.br/cafe</A>. Resources developed in this project provide genetic and genomic tools that may hold the key to the sustainability, competitiveness and future viability of the coffee industry in local and international markets.

11944 downloads

10.

Cloning and sequence analysis of tomato cpDNA fragments: towards developing homologous chloroplast transformation vectors
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Berger, Irving Joseph

; Carraro, Dirce Maria

; Bock, Ralph

; Azevedo, Ricardo Antunes

; Carrer, Helaine

Brazilian Journal of Plant Physiology

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jun 2005, Volume 17 N. 2 Pages 239 - 246

Abstract: Pt En | Text: Pt En | PDF: Pt | PDF: En

Visando o desenvolvimento de vetores homólogos de transformação de cloroplastos para tomate (Lycopersicon esculentum L.), fragmentos cloroplastidiais de tomate cv. IAC-Santa Clara foram clonados e analisados. Isolamento e clonagem de fragmentos PstI/SalI de cloroplastos de tomate deram origem aos plasmídios pIJB1 e pIJB2, contendo fragmentos de 8,6 pb e 6,4 pb respectivamente. Seqüenciamento e análise dos fragmentos revelaram que as seqüências apresentam de 93 a 100 % de identidade com os respectivos fragmentos em tabaco nas regiões codificadoras. Espaços intergênicos mostraram-se menos conservados, sugerindo que divergências evolutivas ocorreram principalmente em regiões não diretamente envolvidas na tradução. A diferença mais marcante encontrada está em um fragmento de 437 pb presente em tabaco na região intergênica entre os genes trnE e trnT, mas ausente no DNA cloroplastidial de tomate. Análise do padrão de restrição das seqüências cloroplastidiais apontou sítios únicos de restrição, sugerindo potencial uso dessas seqüências para a construção de vetores de transformação de cloroplastos. O recente sucesso na expressão de genes exógenos de interesse agronômico em cloroplastos de plantas sugere que essa tecnologia promete revolucionar o conceito e a aplicação da engenharia genética no melhoramento de plantas.

With the view of developing chloroplast transformation vectors based on homologous targeting regions for tomato (Lycopersicon esculentum L.), plastid DNA fragments of tomato cv. IAC-Santa Clara were cloned and analyzed. Isolation and cloning of PstI/SalI chloroplast fragments into the pBlueScript vectors yielded the plasmids pIJB1 and pIJB2 with cpDNA fragments of 8.6 kb and 6.4 kb, respectively. DNA sequencing and computer analyses revealed that the tomato sequences cloned display from 93 to 100 % of identity to the respective fragments in tobacco, which is more pronounced in coding regions. The intergenic spacers are somewhat less conserved suggesting that evolutionary divergences occurred mainly in these putative non-coding regions. The most remarkable difference found is a 437 bp sequence present in tobacco chloroplast genome in the intergenic region of the genes trnE and trnT but completely absent in the tomato chloroplast DNA. Analysis of restriction enzyme digestion patterns revealed several unique restriction sites in the intergenic spacer regions suggesting potential utility of these sequences in species-specific vector construction for tomato chloroplast transformation.

2910 downloads

11.

Seeing the FORESTs for the trees
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Carrer, Helaine

Genetics and Molecular Biology

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2005, Volume 28 N. 3 Pages i - ii

Abstract: En | Text: En | PDF: En

799 downloads Cited 1 time in SciELO

12.

Cloning and expression of cellulase XF-818 of Xylella fastidiosa in Escherichia Coli
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Wulff, Nelson Arno

; Carrer, Helaine

; Pascholati, Sérgio Florentino

Scientia Agricola

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dec 2003, Volume 60 N. 4 Pages 715 - 721

Abstract: Pt En | Text: Pt En | PDF: Pt | PDF: En

Xylella fastidiosa foi a primeira bactéria fitopatogênica que teve seu genoma completamente seqüenciado. A identificação de diversos genes, através de similaridade de seqüências, indicou os possíveis mecanismos de patogenicidade da bactéria. Entretanto, a determinação da função de um gene requer a confirmação experimental e, neste aspecto, a expressão heteróloga é uma poderosa ferramenta. X. fastidiosa coloniza somente o xilema das plantas hospedeiras e a identificação putativa de diversos genes semelhantes a enzimas que degradam a parede celular vegetal, estimularam o presente estudo de catacterização destas enzimas. A clonagem da ORF Xf-818 de X. fastidiosa no vetor de expressão pET20b possibilitou a produção da proteína heterologamente em E. coli. O emprego de IPTG a 0,4 mmol L-1 com 12 h a 32°C, possibilitou as melhores condições para E. coli produzir a proteína heteróloga. Clones de E. coli que expressam Xf-818, apresentam atividade celulásica, degradando eficientemente a celulose. A identificação de Xf-818 como uma endoglicanase foi assim confirmada.

Xylella fastidiosa's genome was the first of a plant pathogen to be completely sequenced. Through comparative sequence analysis many genes were identified and, among them, several potentially involved in plant-pathogen interaction. However, the biological role of each gene should be assigned experimentally. On this regard, heterologous protein expression is a powerful tool to produce proteins from such genes, allowing their characterization. X. fastidiosa lives inside xylem vessels and eventually would degrade pit membranes from xylem cells to move radialy into the host. The identification of several putative plant cell wall degrading enzymes on X. fastidiosa genome prompted the assession of the function of such proteins. The open reading frame (ORF) Xf-818 was cloned into expression vector pET20b and E. coli cells harboring such plasmid exhibited cellulase activity. Using IPTG at 0.4 mmol L-1 with a 12 h incubation at 32°C are the best conditions to produce higher amounts of heterologous protein. The enzyme degrades cellulose confirming the endoglucanase activity of Xf-818.

2948 downloads Cited 1 time in SciELO

13.

Biology, management and biochemical/genetic characterization of weed biotypes resistant to acetolactate synthase inhibitor herbicides
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Monquero, Patrícia Andrea

; Christoffoleti, Pedro Jacob

; Carrer, Helaine

Scientia Agricola

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2003, Volume 60 N. 3 Pages 495 - 503

Abstract: Pt En | Text: Pt En | PDF: Pt | PDF: En

Bidens pilosa e Amaranthus quitensis são as principais plantas daninhas infestantes na cultura de soja [Glycine max L (Merrill)] no Brasil e Argentina, respectivamente. O uso repetitivo de herbicidas inibidores da acetolactato sintase (ALS EC 4.1.3.18) em São Gabriel do Oeste (MS - Brasil) e nas províncias de Córdoba e Tucumã (Argentina), selecionaram biótipos resistentes (R) destas plantas daninhas. Esta pesquisa foi desenvolvida para estudar o manejo, crescimento, a bioquímica e genética destes biótipos resistentes. Em um experimento de campo concluiu-se que chlorimuron-ethyl e imazethapyr (inibidores da ALS), aplicados nas doses recomendadas, não controlaram o biótipo R de B. pilosa, mas os herbicidas alternativos lactofen, fomesafen e bentazon foram eficientes quando aplicados sozinhos ou em mistura com os herbicidas inibidores da ALS. Estudos em casa-de-vegetação confirmaram a resistência cruzada para os biótipos de ambas espécies aos herbicidas dos grupos químicos das imidazolinonas e sulfuniluréias e os herbicidas alternativos sozinhos ou em mistura com os inibidores da ALS controlaram eficientemente populações resistentes e suscetíveis. Análises de crescimento dos biótipos R e S destas plantas daninhas em condições não competitivas mostraram que não existe um custo adaptativo para os biótipos R (efeitos pleiotrópicos). O bioensaio rápido usando inibidores da ALS e ketoacid reductoisomerase (KARI) indicaram que a resistência decorre da insensibilidade da enzima ALS aos herbicidas. Por outro lado, o seqüenciamento do gene que codifica a ALS em R A. quitensis não mostrou mutação no Domínio A, sugerindo que outras posições do gene poderiam estar sofrendo mutações que conferem a insensibilidade da ALS a sulfuniluréias e imidazolinonas.

Bidens pilosa and Amaranthus quitensis are major weeds infesting soybean [Glycine max L (Merrill)] fields in Brazil and Argentina. The repetitive use of acetolactate synthase (ALS EC 4.1.3.18) inhibiting herbicides in São Gabriel do Oeste, MS, Brazil and in the provinces of Córdoba and Tucumã, Argentina, has selected for resistant (R) biotypes of these weeds. Research work was developed to study the management, growth, biochemistry, and genetics of these R weed biotypes. In a field experiment it was found that chlorimuron-ethyl and imazethapyr at recommended rates (both ALS inhibitor herbicides), did not control R B. pilosa, but the alternative lactofen, fomesafen and bentazon were effective, either sprayed alone or mixed with the ALS inhibitor herbicides. Greenhouse studies confirmed the cross-resistance of both R biotypes to the imidazolinone and sulfonylurea herbicides, and these alternative herbicides, when sprayed alone or mixed with the ALS inhibitor, efficiently controlled both R and S populations. A growth analysis of the R and S biotypes of these weeds, under non-competitive conditions, indicated that there is no adaptive cost to the R biotypes (pleiotropic effect). A quick bioassay using ALS and ketoacid reductoisomerase (KARI) inhibitors showed that the resistance of the R biotypes to herbicides is related to a lack of sensitivity of the ALS enzyme to the herbicides. On the other hand, the sequencing of the gene that codifies the ALS resistance in R A. quitensis did not present any mutation in the A Domain region, suggesting that other positions of the gene that confer insensitivity of the ALS to sulfonylurea and imidazolinone herbicides could have mutated.

2283 downloads Cited 3 times in SciELO

14.

Identification of Astyanax altiparanae (Teleostei, Characidae) in the Iguaçu River, Brazil, based on mitochondrial DNA and RAPD markers
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Prioli, Sônia M.A.P.

; Prioli, Alberto J.

; Júlio Jr., Horácio F.

; Pavanelli, Carla S.

; Oliveira, Alessandra V. de

; Carrer, Helaine

; Carraro, Dirce M.

; Prioli, Laudenir M.

Genetics and Molecular Biology

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2002, Volume 25 N. 4 Pages 421 - 430

Abstract: En | Text: En | PDF: En

Astyanax fishes are among the most important food-web components of South America rivers. In the Iguaçu River basin, the Astyanax genus is represented mainly by endemic species. For millions of years, that hydrographic basin has been geographically isolated from the Paraná River basin by the Iguaçu Falls. Recently, a species from the Upper Paraná River basin identified as Astyanax bimaculatus was revised and described as a new species named Astyanax altiparanae Garutti & Britski, 2000. Fauna endemism and geographic isolation triggered interest in investigations to evaluate the identification and genetic relatedness among two A. altiparanae populations from the Upper Paraná River basin and the population identified as A. bimaculatus in the Iguaçu River, upstream from the Iguaçu Falls. Mitochondrial DNA sequences and RAPD markers revealed high genetic diversity within each population, as well as low genetic distance, high gene flow, and high mitochondrial DNA similarity among all three populations. In conjunction with morphological similarities, these results demonstrated that the population presently known as Astyanax bimaculatus in the Iguaçu River should actually be stated as Astyanax altiparanae. Furthermore, it could be inferred that the A. altiparanae population is not endemic and most likely it was recently introduced in the Iguaçu River basin, maintaining the ancestral genetic identity.

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15.

In silico characterization and expression analyses of sugarcane putative sucrose non-fermenting-1 (SNF1) related kinases
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Carraro, Dirce Maria

; Lambais, Marcio R.

; Carrer, Helaine

Genetics and Molecular Biology

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dec 2001, Volume 24 N. 1-4 Pages 35 - 41

Abstract: Pt En | Text: Pt En | PDF: Pt | PDF: En

Quinases de proteínas relacionadas a SNF1 (SnRK) podem desempenhar um papel importante na regulação da expressão gênica em células vegetais. Essa família de proteínas regulatórias é representada pela quinase de proteínas SNF1 (sucrose non-fermenting -1) em Saccharomyces cerevisiae, AMPKs (quinase de proteínas ativadas por AMP) em células de mamíferos e SnRKs (quinase de proteínas relacionadas a SNF1) em células vegetais. A família de SnRKs foi reorganizada em três subfamílias de acordo com suas relações filogenéticas com base nas seqüências de aminoácidos das proteínas. Membros das subfamílias de SnRKs foram identificados em diversas plantas. Existem evidências mostrando que essa família de proteínas está envolvida em resposta a estresses (nutricional e ambiental), apesar de seu papel não ser totalmente compreendido. Nesse trabalho nós identificamos 22 contíguos de ESTs (expressed sequence tags) de cana-de-açúcar codificando SnRKs putativas. O alinhamento das seqüências de aminoácidos das SnRKs putativas de cana-de-açúcar com seqüências de aminoácidos de SnRKs identificadas em outras plantas revelaram um domínio catalítico N-terminal altamente conservado. Além disso, nossos resultados indicaram que em cana-de-açúcar há pelo menos um membro de cada subfamília de SnRKs. Análise do padrão de expressão dos contíguos de EST codificando para SnRK putativas nas 26 bibliotecas selecionadas do banco de dados do Sucest, indicou que membros dessa família de quinases são expressos por toda planta. Membros de cada subfamília não apresentaram padrões de expressão específicos, sugerindo que suas funções não estão correlacionadas com sua relação filogenética, com base nas seqüências de aminoácidos da região N-terminal.

Sucrose non-fermenting-1-related protein kinases (SnRKs) may play a major role in regulating gene expression in plant cells. This family of regulatory proteins is represented by sucrose non-fermenting-1 (SNF1) protein kinase in Saccharomyces cerevisiae, AMP-activated protein kinases (AMPKs) in mammals and SnRKs in higher plants. The SnRK family has been reorganized into three subfamilies according to the evolutionary relationships of their amino acid sequences. Members of the SnRK subfamily have been identified in several plants. There is evidence that they are involved in the nutritional and/or environmental stress response, although their roles are not yet well understood. We have identified at least 22 sugarcane expressed sequence tag (EST) contigs encoding putative SnRKs. The amino acid sequence alignment of both putative sugarcane SnRKs and known SnRKs revealed a highly conserved N-terminal catalytic domain. Our results indicated that sugarcane has at least one member of each SnRK subfamily. Expression pattern analysis of sugarcane EST-contigs encoding putative SnRKs in 26 selected cDNA libraries from the sugarcane expressed sequence tag SUCEST database has indicated that members of this family are expressed throughout the plant. Members of the same subfamily showed no specific expression patterns, suggesting that their functions are not related to their phylogenic relationships based on N-terminal amino acid sequence phylogenetic relationships.

2792 downloads Cited 2 times in SciELO

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