To ensure acceptable survival and initial growth of Eucalyptus grandis clonal and clonal hybrid material planted in the cool temperate and sub-tropical climates of Zululand (KwaZulu-Natal, South Africa) the planting seasons are currently restricted to the winter or summer months respectively. The Zululand coast (sub-tropical climate) experiences traditionally hot and humid summers and as a result Eucalyptus planting is largely restricted to the cool and dry winter months when survival is acceptable (90- 95%). In comparison, the Zululand interior (cool temperate climate) experiences moderately cooler summers followed by drier winters. As a result, the Eucalyptus planting season is reversed to that of the Zululand coast, with most of the planting taking place during the summer months with little or no planting occurring during winter. To ensure adequate transplant survival during and beyond these periods, transplants are planted with large volumes of water at a high cost. To determine whether it was possible to significantly reduce current water volumes at planting and therefore reduce costs, and to potentially extend the current planting seasons, two field trials were initiated during traditionally “unsuitable” planting periods (winter months for the Zululand interior and summer months for the Zululand coast). These trials were established near Kwambonambi on the Zululand coast and at Ntonjaneni in the Zululand interior. Five levels of water were combined with five levels of hydrogel and applied to the pit at planting in a 5 x 5 factorial treatment design for both trials. The tree variates of mortality, height, groundline diameter, crown diameter, corrected leaf surface index (LSIC) and corrected biomass index (BIC) were assessed at regular intervals until the final measurement dates (118 and 128 days after planting for the Ntonjaneni and Kwambonambi trials respectively). For the Kwambonambi trial, the response of transplant survival to the application water was highly significant (p<0,01) 128 days after planting, but not to the application of the hydrogel. Transplant survival nevertheless conformed to the silviculturally accepted norms of 90-95% using the hydrogel, thus water volumes could be significantly reduced without negatively affecting current survival standards. The lack of the expected response of significantly reduced transplant survival to increasing levels of hydrogel could possibly be attributed to the significant rainfall event (146 mm) that fell two days after trial initiation. Increasing levels of both water and hydrogel resulted in significantly enhanced growth (LSIC and BIC: p<0.01) for the final measurement date. For the Ntonjaneni trial, there was a significant (p<0,01) interaction between hydrogel and water, 118 days after planting. There were significant (p<0,01) differences between water only and all hydrogel treatments, with the hydrogel treatments performing significantly better. Optimum transplant survival for water only treatments was 50% using 4000 ml water while that for hydrogel treatments was 100% using 6 g hydrogel with 1000 ml of water and 12 g hydrogel with 2000 ml of water. The variates, corrected leaf surface index and corrected biomass index indicated that tree growth was significantly enhanced by the addition of a hydrogel over all levels of water. A pot trial was subsequently implemented to ascertain whether the significant increases obtained for initial transplant growth for the sandy clay loam soils of Ntonjaneni were due to an initial but unsustainable positive response of the roots to the presence of the hydrogel, or whether root growth was sustainably advantaged by the presence of the hydrogel. There was a highly significant (p<0,01) response of root biomass and above ground biomass over all levels of hydrogel, including a significant positive linear (p<0,01) relationship between increased root biomass and above ground biomass. This clearly indicated that initial root growth was not negatively affected by the addition of the soil-amending hydrogel Stockosorb 400K.

[No abstract submitted] / Science, Faculty of / Botany, Department of / Zoology, Department of / Graduate

One of the phenomena uncovered in cytological studies of Abies grandis LIndley, the Lowland White Fir, was the existence of pairing of chromosomes in somatic cells of the very young ovules. Huskins (1948) has shown that somatic pairing is not rare in the plant world, but is seldom properly recorded. It was thought necessary to undertake a study of the morphology of the Individual chromosomes of the tree. Counts and measurements were made on the chromosomes in several cells, and the measurements reduced to a percentage of the total chromosome length in the cell. Positions of the spindle-fibre attachments or centromeres were also tentatively located and recorded as percentages. The lengths of the twelve chromosomes in the genome are as follows: Chromosome 1, 14.4% units long; 2, 11.3% units; 3, 10.6% units; 4, 9.6% units; 5, 9.1% units; 6, 8.4% units; 7, 8.1% units; 8, 1.2% units; 9, 6.4% units; 10, 6.1% units; 11, 5.5% units; 12, 3.3% units. Two chromosomes have club-shaped ends without any constriction, while five have single terminal knobs. One has three constrictions, one has two, and four have but a single constriction. One chromosome is dicentric, four are approximately isomeral, five are distinctly heteromeral, and two have terminal centromeres. Somatic cells from young ovules showing apparent pairing of chromatin strands at early anaphase were examined. In one cell studied, the pairing is so distinct and the similarity between chromatin threads so striking that It is thought to be indication of some sort of reduced meiosis. The differences between somatic pairing and true meiosis are discussed and also some theories on the reason for somatic pairing. Some problems in technique of conifer cytology and the methods used in this study are set forth. / Science, Faculty of / Botany, Department of / Graduate

Dissertation submitted in compliance with the requirements for the Master's Degree in Technology: Biotechnology, Technikon Natal, 2001. / Biotechnology techniques such as micropropagation VIa somatic embryogenesis offer potential significant advances in the improvement of forest species, which could sustain forest production in South Africa, as well as globally, without increased use of land. In order to apply such techniques to commercial breeding and clonal programmes of E. grandis species, it is necessary to develop reliable and efficient protocols applicable to explants of proven superior genotypes. Most of the research on E. grandis somatic embryogenesis has used the genetically variable embryos or seedlings as explant sources, which results in the propagation of material of unproven genetic value. In order to exploit somatic embryogenesis maximally for cloning of superior trees, somatic embryos have to be induced from highly selected and, hence, mature trees. The aim of this investigation was to develop such a protocol for E. grandis and to test its applicability to various E. grandis hybrids. Somatic embryos were induced from buds, stems, leaves and petioles, with petioles and buds giving the best results. Thus, these were selected for further studies which involved testing the effect of medium composition on embryogenic callus induction. Media used for this purpose contained MS or B5 nutrients, 1 mg.l' 2,4-D, 0.5 g.r! glutamine, 0.5 g.r! casein hydrolysate, 4 g.r! Gelrite and 30 or 50 g.rl sucrose. All the media tested were able to support induction of embryogenic callus, although the number of explants producing embryogenic calli was affected significantly by the media composition (10-91 %). Callus induction media with B5 nutrients seemed to have a significant effect onn the developmental stage of embryos in the callus induction medium. Presence of 50 g.r! sucrose in the callus induction medium reduced the embryo yield, but the progress of embryo development was enhanced. The callus induction medium containing B5, 1 mg.l' 2,4-D, 0.5 g.rl glutamine, 0.5 g.r! casein hydrolysate, 4 g.r! Gelrite and 30 g.l' sucrose was chosen for subsequent studies. Of all the media tested for embryo development, the medium with B5, 2.5 mg.l' 2iP, 0.5 g.r! glutamine, 0.5 g.r! casein hydrolysate, 4 g.r! Gelrite and 50 g.r! sucrose was found to be the most suitable for embryo development to the cotyledonary stage. Experiments involving incorporation of both ABA and 2iP aiming at maturation of E. grandis somatic embryos led to an increase in size of the cotyledonary embryos formed but not to germination. / M

Os isoprenóides são compostos essenciais a todos os organismos e representam um grupo extremamente amplo estruturalmente e funcionalmente. Em plantas, mais de 30.000 compostos desta classe foram identificados até hoje. Todas as plantas produzem isoprenóides que desempenham papéis essenciais como os carotenóides, as clorofilas e as plastoquinonas (fotossíntese); ubiquinona (respiração); citocininas, brassinosteróides, giberelinas, ácido abscísico (regulação do crescimento e desenvolvimento). No entanto, a maior variedade de isoprenóides é representada pelos metabólitos secundários (óleos essenciais como eucaliptol, cineol, citronelal). Os isoprenóides possuem papel marcante nas relações da planta com o ambiente onde estão inseridas, já que medeiam as relações planta-inseto, planta-microorganismo e planta-planta. Devido ao valor comercial dos compostos isoprenóides, há grande interesse em produzi-los por bioengenharia em bactérias ou em plantas e o entendimento do papel dos genes e proteínas codificadas na rota de síntese dessas unidades básicas de formação de terpenóides é extremamente importante. Nesse trabalho, nós recuperamos, a partir da seleção dos plasmídios de clonagem pSPORT1 (Invitrogen) purificados do Projeto “Genolyptus: Rede Brasileira de Pesquisa do Genoma de Eucalyptus”, os clones selecionados e potencialmente codificadores de 1-desoxi-D-xilulose-5-fosfato redutoisomerase (DXR) e de outras enzimas atuantes nas rotas de síntese de isoprenóides, como 1-desoxi-D-xilulose 5-fosfato sintase (DXS), mevalonato difosfato descarboxilase (MDC), isopentenil difosfato isomerase 1 (IPPI1) e isopentenil difosfato isomerase 2 (IPPI2). Nos estoques de plasmídios do projeto Genolyptus, foi possível encontrar as sequências completas dos genes dxr, ippi1 e ippi2. Os genes foram analisados in silico e a sequência de ippi1 foi utilizada na modelagem molecular e dinâmica molecular para avaliação de características peculiares do folding protéico desse tipo de proteína eucariótica. Os genes foram clonados em vetores pGEX-4T (GE Healthcare) e heterologamente expressos em Escherichia coli. Foi realizada, também, uma análise transcricional comparativa dos genes selecionados pela técnica de microarranjos. / Isoprenoids are essential to all organisms and are the most structurally and functionally diverse group of plant metabolites. In plants, more than 30,000 compounds of this class were identified to date. All plants produce isoprenoids that can play essential roles as carotenoids, chlorophylls and plastoquinone (photosynthesis); ubiquinone (respiration); regulation of growth and development (cytokinins, brassinosteroids, gibberellins, abscisic acid). However, the majority of isoprenoids is represented by secondary metabolites (essential oils like eucaliptol, cineol, citronelal). Isoprenoids have important roles in the relationships between plants and the environment, since they can mediate plant-insect, plant-microorganism and plant-plant interactions, as well as participate in abiotic stress responses. Due to the high value of isoprenoid compounds, there is great interest in producing them by bioengineering in bacteria or plants. Understanding the role of genes and proteins related to the biosynthetic pathway of isoprenoids is extremely important. In this work, we retrieved, from the collection of cloning plasmids pSPORT1 (Invitrogen) generated in the “Genolyptus Project: The Brazilian Research Network on the Eucalytpus Genome”, selected clones that potentially codified the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and other enzymes from the isoprenoid biosynthetic pathway including 1-deoxy-D-xylulose 5-phosphate synthase (DXS), mevalonate disphosphate decarboxilase (MDC), isopentenil disphosphate isomerase 1 (IPPI1) and isopentenil disphosphate isomerase 2 (IPPI2). Complete sequences of the dxr, ippi1 and ippi2 genes were succesfully recovered from the Genolyptus stocks. The genes were analyzed in silico and the ippi1 sequence was used in studies of molecular modeling and molecular dynamics in order to evaluate specific folding characteristics of this kind of eukaryotic IPPI. The genes were cloned into pGEX-4T vectors (GE Healthcare) and expressed in Escherichia coli. Also, the transcriptional analysis of the selected genes was performed by microarray analysis.

A indústria de base florestal é estratégica para o Brasil devido ao seu perfil fortemente exportador. O setor responde pela segunda posição na balança comercial do agronegócio brasileiro, ficando atrás somente da soja em grão. Atualmente, a área ocupada com florestas de eucaliptos no Brasil atinge 1,9 milhões de ha. Diante da importância sócio-econômica que a silvicultura desempenha no mercado brasileiro e do aumento progressivo das áreas plantadas com florestas de Eucalyptus, o grande desafio para o melhoramento do eucalipto está na integração da biotecnologia mais avançada ao seu cultivo, o que compreende a identificação de genes controladores das características de importância econômica e ambiental e a transferência destes genes entre árvores por meio de cruzamentos controlados ou modificação direcionada. Portanto, os objetivos deste estudo foram apresentados em 3 capítulos distintos: I - a identificação de genes expressos em flores de E. grandis em processo de antese; II - o estudo mais refinado de mineração e identificação de genes potencialmente codificadores de fatores de transcrição presentes no genoma de E. grandis; e III - seleção de 50 genes cujas expressões mostraram-se constitutivas entre folhas e xilemas de E. grandis e xilema de E. globulus, pela técnica de hibridização de microarranjos de DNA. No capítulo I é apresentada uma breve fundamentação teórica sobre as flores de E. grandis e a importância do estudo da expressão gênica e da identificação de genes envolvidos em determinados processos metabólicos e fisiológicos das plantas. Nos resultados, apresentados juntamente com a discussão, estão apresentados o conjunto de transcritos identificados e a anotação dos mesmos conforme as bibliotecas geradas. As sequências de genes expressos estão fundamentalmente envolvidas na manutenção do órgão, na senescência e em respostas a estímulos ambientais. Também são mostrados resultados obtidos por RT-qPCR para genes selecionados a partir das anotações cujo perfil de transcrição foi avaliado para as partes da flor, folha e xilema. Ao final do capítulo, é feita uma breve descrição da metodologia e das conclusões referente a este estudo. A partir dos dados anotados dos genes expressos nas bibliotecas de flores e botões florais descritos no primeiro capítulo, foram encontradas algumas famílias de fatores de transcrição, dentre elas, a família Dof, encontrada nas bibliotecas de carpelos/receptáculos florais. Assim, um segundo capítulo foi redigido na forma de manuscrito de artigo científico a ser submetido ao periódico BMC Plant Biology, em língua inglesa. Após a fundamentação teórica sobre os fatores Dof em plantas, foram apresentados os resultados obtidos de um estudo mais refinado de mineração e identificação de genes potencialmente codificadores destes fatores de transcrição presentes no genoma de E. grandis. Posteriormente, uma quantificação dos níveis de mRNA para alguns dos genes Dof de E. grandis a partir da técnica de RT-qPCR foi realizada. A análise foi realizada para órgãos diferentes da planta e em plântulas submetidas a estresses abióticos e sinalização por reguladores de crescimento vegetais. Os resultados e a discussão deste capítulo também são mostrados em uma única sessão, assim como a descrição da metodologia e as conclusões sobre esta etapa. O terceiro e último capítulo, também apresentado na forma de manuscrito de artigo científico a ser submetido ao periódico BMC Plant Biology, em língua inglesa, é composto de um estudo paralelo aos temas citados acima, realizado a partir da seleção de 50 genes cujas expressões mostraram-se constitutivas entre folhas e xilemas de E. grandis e xilema de E. globulus, pela técnica de hibridização de microarranjos de DNA. Dos 50 genes selecionados e anotados, oito foram selecionados para a validação por RT-qPCR em seis espécies de Eucalyptus e em três órgãos diferentes (flor, folha e xilema). As expressões destes genes candidatos foram comparadas àquelas de sete genes normalizadores tradicionais, usados frequentemente em estudos de avaliação da expressão gênica em plantas. Como nos demais capítulos, as conclusões referentes a este estudo, bem como a metodologia empregada, são apresentadas ao final do capítulo. Na parte final desta tese, constam as conclusões gerais sobre a totalidade do trabalho conduzido. / The forest industry is strategic to Brazil due to its strong export profile. The sector accounts for the second position in the trade brazilian agribusiness balance. Currently, the area planted with Eucalyptus forests in Brazil reached 1.9 million/ha. Given the socioeconomic importance that forestry plays in the brazilian market and the gradual increase in areas planted with Eucalyptus forest, the great challenge for the Eucalyptus breeding is the integration of advanced biotechnology to cultivation, which includes the identification of economic importance genes controlling environmental effects and transfer of genes between trees through intersections controlled or directed modification. The objectives of this study were presented in three distinct chapters: I - the identification of expressed genes in E. grandis flowers in the anthesis process; II – more refined study, mining and identification of genes potentially encoding transcription factors present in the E. grandis genome and III - selection of 50 genes whose expressions were shown to be constitutive in E. grandis xylem and leaves and E. globulus xylem, by DNA microarray hybridization tecnique. In Chapter I, we present a brief theoretical background on the E. grandis flowers and the importance of studying gene expression and identifying genes involved in metabolic and physiological processes of plants. The results, presented transcripts sets identified and annotation the libraries generated. The sequences of expressed genes are mainly involved in organ maintaining, senescence and in responses to environmental stimuli. Also shown are results obtained by RT-qPCR for selected genes from the notes whose transcription profile was assessed for part of the flower, leaf and xylem. At the end of the chapter is a brief description of the methodology and conclusions about this study. From the data recorded in the libraries of genes expressed in flowers and flower buds described in the first chapter, we found some families of transcription factors, among them, the Dof family, found in the libraries of carpel/floral receptacles. Thus, a second chapter was drafted as a manuscript of a scientific paper to be submitted to the journal BMC Plant Biology. After the theoretical DOF on the factors in plants, we presented the results of a more refined mining and identification of genes potentially encoding these transcription factors present in the E. grandis genome. Subsequently, a quantification of mRNA levels for some of E. grandis Dof genes from the RT-qPCR was performed. The analysis was performed for different plant organs and in seedlings subjected to abiotic stress signaling and plant growth regulators. The results and discussion of this chapter are also shown in a single session, as well as a description of the methodology and conclusions about this step. The third and final chapter, also presented as a manuscript of a scientific paper to be submitted to the journal BMC Plant Biology, is composed of a parallel study to the subjects mentioned above, performed by the selection of 50 genes whose expression showed is constitutive of E. grandis leaves and xylem and E.globulus xylem, by the DNA microarray hybridization tecnique. Of the 50 selected genes and annoted, eight were selected for validation by RT-qPCR in six Eucalyptus species and three different organs (flower, leaf and xylem). The expression of these candidate genes were compared to those of traditional standard-seven genes, often used in evaluation studies of gene expression in plants. As in other chapters, the findings of this study and the methodology employed, are included at the end of the chapter. In the final part of this thesis, contains the general conclusions about the totality of the work conducted.

O algodoeiro sofre o ataque de diversos insetos-praga, sendo o principal o bicudodo- algodoeiro (Anthonomus grandis). Visando alternativas mais eficazes para o controle de insetos, tem-se buscado a transgenia de plantas, por meio da introdução de genes que codificam moléculas entomotóxicas. Entre as diferentes estratégias de engenharia genética utilizadas para controle de insetos-praga, a estratégia envolvendo o silenciamento gênico, pelo uso da interferência mediada por RNA (RNAi), é atualmente bastante promissora e de grande relevância. O trabalho aqui apresentado resultou na geração de uma biblioteca completa de genes expressos de A. grandis, por meio de sequenciamento completo de ESTs, obtidas a partir de diferentes estágios do desenvolvimento do inseto. O pirosequenciamento do transcritoma de A. grandis gerou mais de 500.000 reads e um banco de dados com 20.841 contigs com alta qualidade. Após a montagem e anotação das sequências foram realizadas buscas por genes essenciais do inseto, candidatos ao silenciamento por RNAi. Um desses genes, que codifica a enzima lacase2, foi selecionado e avaliado, mostrando alto potencial para o controle do bicudo do algodoeiro. Esta enzima faz parte do processo de formação e esclerotização da cutícula do inseto. Sua expressão se mostrou quantitativamente maior nas fases mais tardias do ciclo de vida do inseto. Observou-se que a expressão relativa dos transcritos de lacase2 foi altamente reduzida e o desenvolvimento do inseto foi afetado pelo silenciamento gênico. Além da validação do gene de lacase2 para potencial controle do bicudo, este trabalho disponibiliza dados para a escolha de genes de referência para experimentos de quantificação relativa de transcritos por PCR em tempo real em várias partes de larvas e adultos de A. grandis. O conjunto de dados aqui obtidos poderá contribuir enormemente para o agronegócio brasileiro e será de grande importância para a obtenção e geração de eventos de algodão transgênicos resistentes ao bicudo do algodoeiro. / Cotton plants are subjected to the attack of several insect pests. In Brazil, the cotton boll weevil Anthonomus grandis is the most important cotton pest. The use of transgenic plants by introducing genes coding for entomotoxic molecules is one of the recent efficient approaches used in plant pest control. Among those, the use of gene silencing by interference RNA (RNAi) as a technique for insect control is very promising. The work presented in the next pages shows the use of pirosequencing technique for the construction of a cDNA library formed by A. grandis expressed genes in all developmental stages of the insect. The pirosequencing of A. grandis transcriptome resulted in more than 500,000 reads and a data set of high quality 20,841 contigs. After sequence assembly and annotation, the library was screened for insect essential candidate genes for RNAi-mediated gene silencing. One gene, coding for the enzyme laccase2 was selected and has demonstrated high potential for insect control. This enzyme is involved in insect cuticle formation and sclerotization, and the gene was found to be more expressed in the insect pupa and adult developmental stages. Insect development was affected by gene silencing and the relative expression of laccase2 transcripts was highly decreased. Moreover, this work provides data for choosing reference genes to be used in qPCR experiments for several parts of boll weevil larvae and adult organism. The results provided in this work may contribute to Brazilian agribusiness by the potential important generation of genetically modified cotton plants with significant resistance to cotton boll weevil.

As proteínas-cinases ativadas por mitógenos (MAPKs) participam de rotas de transdução de sinais universais em eucariotos e compõem cascatas de fosforilação que levam as células a responder a estímulos extracelulares. Em plantas, MAPKs estão associadas a processos de desenvolvimento e na reposta a hormônios e a estresses bióticos e abióticos. Em trabalhos anteriores de nosso grupo, o gene Egmpk5 de Eucalyptus grandis foi clonado e seu padrão de expressão foi caracterizado em plantas submetidas a diferentes tratamentos. Ainda, plantas de Nicotiana tabacum SR1 foram transformadas com Egmpk5 sob regulação do promotor CaMV 35S para futuro estudo de função do gene. No presente trabalho, foi avaliada mais amplamente a estrutura, a expressão e o produto deste gene. Análises in silico revelaram que Egmpk5 apresenta-se como cópia única no genoma, está organizado em seis éxons e seus cinco íntrons possuem sítios canônicos de splicing de RNA. O produto deduzido deste gene apresenta em sua sequência a assinatura do domínio proteína-cinase e os motivos de ligação a ATP e de ativação/dupla fosforilação, indicando a possibilidade de ser uma proteína funcional. Árvore filogenética foi construída pelo método de Máxima Verossimilhança utilizando o algoritmo MUSCLE para alinhamento das sequências e um valor de bootstrap de 500 repetições com sequências peptídicas similares à proteína deduzida EgMPK5 e permitiu a identificação de MAPKs de outras plantas com função já comprovada na sinalização da divisão celular e na resposta a estresses abióticos, ferimento e patógenos. Análise de RT-qPCR mostrou a expressão de Egmpk5 em níveis comparáveis em raízes, caules e folhas de plantas de E. grandis tratadas com água, e foi detectado um aumento de sua expressão em folhas tratadas com ácido abscísico (ABA) e em todos os órgãos tratados com solução de cloreto de sódio a 200 mM. O estudo da região promotora de Egmpk5 revelou um grupo de possíveis elementos de ação cis relacionados a respostas a estresses. Seis linhagens transformadas de tabaco que tiveram seu estado transgênico confirmado por PCR foram desafiadas com tratamentos de seca e cloreto de sódio, e linhagens mais tolerantes foram identificadas. Ao final destas diversas análises, os resultados obtidos sugerem a participação de Egmpk5 em respostas a estresses abióticos. / Mitogen-activated protein kinases (MAPKs) are part of phosphorylation cascades found in all eukaryotes and are responsible for transducing extracellular stimuli into intracellular responses. In plants, MAPK cascades are involved in growth and development processes and have roles in the response to hormones and biotic and abiotic stresses. In our previous studies, the Egmpk5 gene from Eucalyptus grandis was cloned and its expression profile was characterized in plants subjected to different treatments. Also, Nicotiana tabacum SR1 plants were transformed with Egmpk5 under the control of the CaMV 35S promoter for further function studies. In the present study the structure, expression profiles and product of this gene were further characterized. In silico analyses revealed that the Egmpk5 gene is present as a single copy in the E. grandis genome, and that its structure comprises six exons and five introns with conserved sites for transcript splicing. The deduced product of this gene presents the protein kinase domain signature, ATP-binding site and activation/dual phosphorylation motifs in its structure. A phylogenetic tree was built with sequences similar to the deduced EgMPK5 peptide and lead to the identification of plant MAPKs that have had proven activity in cell division signaling and in the response to abiotic stresses, wounding and pathogens. RT-qPCR analysis showed equal expression of Egmpk5 in roots, stems and leaves of plants treated with water and we detected an increase of expression in leaves treated with abscisic acid (ABA) and also increase in expression in all three organs upon treatment with 200 mM sodium chloride. Analysis of the promoter region of Egmpk5 revealed a group of putative cis-acting elements related to stress responses. Six transformed tobacco lines were confirmed by PCR and were assayed with drought and sodium chloride treatments for the identification of more tolerant individuals. After all analyses performed, results allowed us to suggest that Egmpk5 is involved in abiotic stresses responses.

No description available.

Os isoprenóides são compostos essenciais a todos os organismos e representam um grupo extremamente amplo estruturalmente e funcionalmente. Em plantas, mais de 30.000 compostos desta classe foram identificados até hoje. Todas as plantas produzem isoprenóides que desempenham papéis essenciais como os carotenóides, as clorofilas e as plastoquinonas (fotossíntese); ubiquinona (respiração); citocininas, brassinosteróides, giberelinas, ácido abscísico (regulação do crescimento e desenvolvimento). No entanto, a maior variedade de isoprenóides é representada pelos metabólitos secundários (óleos essenciais como eucaliptol, cineol, citronelal). Os isoprenóides possuem papel marcante nas relações da planta com o ambiente onde estão inseridas, já que medeiam as relações planta-inseto, planta-microorganismo e planta-planta. Devido ao valor comercial dos compostos isoprenóides, há grande interesse em produzi-los por bioengenharia em bactérias ou em plantas e o entendimento do papel dos genes e proteínas codificadas na rota de síntese dessas unidades básicas de formação de terpenóides é extremamente importante. Nesse trabalho, nós recuperamos, a partir da seleção dos plasmídios de clonagem pSPORT1 (Invitrogen) purificados do Projeto “Genolyptus: Rede Brasileira de Pesquisa do Genoma de Eucalyptus”, os clones selecionados e potencialmente codificadores de 1-desoxi-D-xilulose-5-fosfato redutoisomerase (DXR) e de outras enzimas atuantes nas rotas de síntese de isoprenóides, como 1-desoxi-D-xilulose 5-fosfato sintase (DXS), mevalonato difosfato descarboxilase (MDC), isopentenil difosfato isomerase 1 (IPPI1) e isopentenil difosfato isomerase 2 (IPPI2). Nos estoques de plasmídios do projeto Genolyptus, foi possível encontrar as sequências completas dos genes dxr, ippi1 e ippi2. Os genes foram analisados in silico e a sequência de ippi1 foi utilizada na modelagem molecular e dinâmica molecular para avaliação de características peculiares do folding protéico desse tipo de proteína eucariótica. Os genes foram clonados em vetores pGEX-4T (GE Healthcare) e heterologamente expressos em Escherichia coli. Foi realizada, também, uma análise transcricional comparativa dos genes selecionados pela técnica de microarranjos. / Isoprenoids are essential to all organisms and are the most structurally and functionally diverse group of plant metabolites. In plants, more than 30,000 compounds of this class were identified to date. All plants produce isoprenoids that can play essential roles as carotenoids, chlorophylls and plastoquinone (photosynthesis); ubiquinone (respiration); regulation of growth and development (cytokinins, brassinosteroids, gibberellins, abscisic acid). However, the majority of isoprenoids is represented by secondary metabolites (essential oils like eucaliptol, cineol, citronelal). Isoprenoids have important roles in the relationships between plants and the environment, since they can mediate plant-insect, plant-microorganism and plant-plant interactions, as well as participate in abiotic stress responses. Due to the high value of isoprenoid compounds, there is great interest in producing them by bioengineering in bacteria or plants. Understanding the role of genes and proteins related to the biosynthetic pathway of isoprenoids is extremely important. In this work, we retrieved, from the collection of cloning plasmids pSPORT1 (Invitrogen) generated in the “Genolyptus Project: The Brazilian Research Network on the Eucalytpus Genome”, selected clones that potentially codified the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and other enzymes from the isoprenoid biosynthetic pathway including 1-deoxy-D-xylulose 5-phosphate synthase (DXS), mevalonate disphosphate decarboxilase (MDC), isopentenil disphosphate isomerase 1 (IPPI1) and isopentenil disphosphate isomerase 2 (IPPI2). Complete sequences of the dxr, ippi1 and ippi2 genes were succesfully recovered from the Genolyptus stocks. The genes were analyzed in silico and the ippi1 sequence was used in studies of molecular modeling and molecular dynamics in order to evaluate specific folding characteristics of this kind of eukaryotic IPPI. The genes were cloned into pGEX-4T vectors (GE Healthcare) and expressed in Escherichia coli. Also, the transcriptional analysis of the selected genes was performed by microarray analysis.