No description available.

In most mammals, sexual fate is determining genetically by the presence of the SRY gene which encoded the testis-determining factors on the Y chromosome. Likewise, avian sex is determined genetically. At day 3.5 (stage 22; HH) in chicken embryogenesis, the gonadal primordium begins forming. Thus, to identify the novel sex-determinating genes in early chicken embryos, subtractive cDNA libraries from male-minus-female (M-F) and female-minus-male (F-M) of 3 Dpc. embryos were established. Both collected male and female chicken total mRNAs were purified using Dynabeads. After a blund-end restriction endonuclease Rsa I digestion of cDNA, adaptor ligation for tester cDNA was performed. When first and second cDNA hybridization was finished, those nonredundant cDNA between tester and driver will be amplified by two rounds of PCR. Subsequently, TA-cloning was performed and the cDNA fragments were PCR-amplified using M13 primers. PCR products of Clones were first screened by differential screening hybridization to decrease false positive inserts. Then, gene annotation was carried out by data-mining in public databases, GeneBank (NCBI, USA). Finally, 40 known and 71 novel transcripts of M-F cDNA library, 88 known and 128 novel transcripts of F-M cDNA library were identified. In M-F subtracted library, 4 identified known genes were located on Z sex chromosome such as WD repeat domain 36 (WDR36), PC4 and SFRS1 interacting protein 1 (PSIP1), serum response factor binding protein 1 (SRFBP1) and glycine dehydrogenase (decarboxylating) (GLDC). Another two identified known genes, laminin alpha 1 (LAMA1) and leukocyte cell derived chemotaxin 1 (LECT1) were reported be relate to cell differentiation and development. In F-M subtracted library, only Wpkic-8 was located on W sex chromosome. Other identified genes like slowmo homolog 2 (Drosophila) (SLMO2), collagen, type IV, alpha 1 (COL4A1), anterior gradient 2 homolog (Xenopus laevis), transcript variant 2 (AGR2), solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 6 (SLC25A6) and prolyl endopeptidase (PREP) were also found expressed higer in human ovary then testis. PREP was proposed that it may play a role in mediating sperm death by regulating the levels of thyrotropin-releasing hormone analogs and in mediating sperm death associated with necrozoospermia. These transcripts located on W or Z sex chromosome identified from subtracted libraries may play an important role in sex determination mechanism.

A pipeline was developed for the quantitative screening and sequence management of clones from suppression subtractive hybridization (SSH) cDNA libraries. The pipeline is particularly useful for gene discovery in non-sequenced organisms, and was illustrated with SSH library data from pearl millet (Pennisetum glaucum) and cowpea (Vigna unguiculata) and Arabidopsis (Arabidopsis thaliana) ecotype Kil-0. The objective of each library was to identify stress-response genes. cDNA microarrays provide a high-throughput screening method. Accordingly, these SSH libraries were amplified by PCR and spotted onto glass microarray slides. Subtracted and un-subtracted cDNA samples, that were used to construct the SSH libraries were prepared as Cy3- and Cy5-labeled targets and hybridized to the microarrays. The R package SSHscreen version 2.0.0, available from http://microarray.up.ac.za/SSHscreen/, was developed to analyze the resulting microarray data using limma (linear models for microarray data) functions. Commonly, loess normalization is used for within-slide normalization, however this is based on the assumption that most of the genes on the array are not differentially expressed. This is legitimate for most whole genome microarray experiments, however it is not appropriate when the array is constructed from an SSH library which is enriched for differentially expressed genes. Therefore, control spot-based normalization was used in the SSHscreen analysis. Empirical Bayes methods were employed to calculate the moderated t-statistic using functions from the limma package. This procedure in effect borrows information from the ensemble of genes to aid with inference about individual genes, taking advantage of the parallel structure whereby the same model is fitted to the data for each gene. In the Arabidopsis, pearl millet and cowpea forward libraries, 18%, 58% and 58% of the clones were identified as significantly up-regulated (adjusted p-value < 0.05) and in the reverse libraries, 18%, 30% and 28% significantly down-regulated, respectively. SSHscreen analysis was used to assist in selection of clones for sequencing. The SSHscreen data output (ranked gene lists in terms of differential expression), as well as the selected sequences in FASTA format were uploaded to SSHdb. For the Arabidopsis library, 114 out of the 262 sequenced clones (55%) were identified as unique/non-redundant; and for the pearl millet and cowpea libraries respectively, 37% and 33% of the sequenced clones were unique. SSHdb was developed as a web-based tool for sequence management and annotation of clones in SSH libraries and can freely be accessed at http://sshdb.bi.up.ac.za. BLAST analysis that was carried out when sequences were uploaded to SSHdb was used to combine clones with the same sequence into redundant partner groups, as well as identify putative annotations for each group. Individual clones from the abovementioned SSH libraries were selected and an independent technique, quantitative PCR, was used to validate the microarray/SSHscreen results. The pipeline was applied successfully to Arabidopsis, pearl millet and cowpea SSH cDNA libraries. Interesting genes in each case were identified for further study. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted

O splicing alternativo é o processo pelo qual diversos transcritos podem ser gerados a partir de um único gene, sendo de extrema importância para diversidade do repertório transcricional e proteico. Diferentes variantes de splicing são expressas entre os diferentes tecidos e estágios de desenvolvimento garantindo o funcionamento normal da célula, portanto, qualquer alteração neste padrão pode resultar no aparecimento de doenças. Neste contexto, o objetivo deste trabalho foi o estabelecimento de metodologias para identificação de variantes de splicing em câncer de mama sob influência do oncogene ERBB2, o qual é um marcador de mau prognóstico altamente expresso em cerca de 30% dos tumores de mama. Foram estabelecidas duas estratégias para construção de bibliotecas de cDNA. A construção de bibliotecas de cDNA enriquecidas para splicing alternativo, baseada na formação e captura de moléculas de heteroduplexes em combinação com a amplificação de RNAm, foi realizada a partir de RNA total de linhagem celular de mama e a partir de um grupo de cinco amostras tumorais, todas com alta expressão de ERBB2. Foram identificadas 79 possíveis variantes de splicing alternativo em câncer de mama, das quais 18 foram selecionadas para validação por RT-PCR. Foi obtida uma taxa de vallidação de 94% e foram identificadas duas novas variantes de splicing alternativo. A regulação da expressão mediada por ERBB2 de três variantes de splicing foi confirmada por duas metodologias distintas, eletroforese em chip e estratégia baseada na ligação de sondas específicas, que revelou desbalanço de expressão entre as variantes, demonstrando a influência do oncogene na regulação de variantes de splicing. A segunda abordagem utilizada, foi a construção de bibliotecas de cDNA para avaliação do transcriptoma total, utilizando sequênciamento de alto desempenho. Foram utilizados RNA total de duas linhagens celulares de mama que diferem apenas na expressão do gene ERBB2. Foram identificadas 2.865 novas variantes de splicing, das quais 20, que reportaram a identificação de um novo éxon, foram selecionadas para validação, com uma taxa de validação de 90%. Seis destas variantes apresentaram aumento de expressão na linhagem com alta expressão de ERBB2. Além disso, foi detectado um enriquecimento de algumas categorias de variantes na linhagem celular com alta expressão de ERBB2, reforçando a influência do oncogene na regulação do splicing alternativo, podendo resultar em variantes de splicing associadas a este grupo de câncer de mama, que podem ser candidatas a marcadores moleculares. / Alternative splicing is a process, by which many differente transcripts can be generated by one single gene, significantly expanding the transcriptional and proteomic diversity. Different splicing variants are generated among different transcripts and developmental stages, assuring normal cell function. Therefore, alterations in the splicing process can lead to diseases outcome. In this context, the aim of this study was the establishment of methodologies for the identification of alternative splicing in breast cancer influenced by ERBB2 oncogene, which is a poor prognostic molecular marker, highly expressed in 30% of human breast cancer. Two strategies were established for the construction of cDNA libraries. Alternative enriched splicing libraries, based on heteroduplex capture combined with mRNA amplification, were constructed from total RNA from a cell line and also from five tumor samples, all of them presenting high ERBB2 expression. Seventy nine putative splicing variants were identified and 18 of them were selected for RT-PCR validation. A high validation level was obtained (94%) and two novel alternative splicing variants were identified. ERBB2 mediated regulation was confirmed for three variants by two distinct methodologies, electrophoresis on a chip and probe specific ligation approach. The alteration in the expression balance of variants suggests the influence of the oncogene in the splicing pattern regulation. The second strategy was the construction of cDNA libraries for global transcriptome analysis based on deep sequencing. Total RNA from two mammary epithelial cell lines expressing different ERBB2 levels were used and 2,865 novel splicing variants were identified. Twenty novel events reporting the inclusion of novel exons were selected for RT-PCR validation with 90% validation rate. Six variants presented higher expression in the cell line with high levels of ERBB2. Moreover enrichment in splicing events was detected in the ERBB2 high expressing cell line, supporting the ERBB2 influence in alternative splicing regulation, possibly resulting in splicing variants associated to this subgroup of cancer that can be tested as molecular markers.

Este trabalho mostrou a purificação de uma apirase em extratos de tegumento de vermes adultos de S. mansoni com razão de hidrólise de ADP/ATP aproximadamente 2. Análise da amostra por MALDI-TOF evidenciou a presença de helicase e carboxilesterase, sendo que a atividade de hidrólise de ADP ainda não foi descrita para estas proteínas. Através de \"Western blot\" mostrou-se que a proteína purificada não tem epítopos reconhecíveis por anticorpo anti-apirase de batata, e que a imunoreatividade cruzada com apirase da família CD39 está presente na fração insolúvel que não foi utilizada no processo de purificação. Esta tese caracteriza também a utilização de \"primers\" consenso degenerados na construção de bibliotecas de cDNA com PCR de baixa estringência. A introdução do \"touch-down\" PCR aumentou o número médio de sequências não redundantes por biblioteca de 20 para 40. O \"touch-down\" associado ao uso de primers híbridos consenso-degenerados (40 pb) elevou a média de sequências não redundantes para 300. Este número significa uma diminuição de cerca de 10 vezes na quantidade de trabalho de clonagem e sequenciamento necessário para se gerar o mesmo montante de dados (Fietto et al., 2000, addendum patent application Nº196,716). A diminuição da concentração de sal na reação e abaixamento da velocidade de rampa entre os passos de anelamento e extensão gerou os melhores perfis de amplificação de cDNA. Bibliotecas feitas com primers híbridos mais curtos (25 pb) mostraram-se mais redundantes, refletindo uma maior especificidade por certas sequências alvo. Com estes primers, usando-se RNAm de tegumento, clonamos um fragmento parcial de cDNA altamente homólogo à CD39 de camundongo. Esta sequência serviu como base para o isolamento de grande parte do gene de Schistosoma (2,9 Kb) através de RACE-PCR (Rapid Amplification of cDNA Ends). / This work shows purification of an apyrase from the tegument membrane of S. mansoni adult worms that exhibited an ADP/ATP hydrolysis ratio near 2. MALDI-TOF analyses showed that helicase and carboxylesterase were present in the sample, however in the literature there is no ADPase activity described for either enzyme. Western blot analyses showed that the purified apyrase ha no epitopes common to members of the CD39 apyrase family. On the other hand, a CD39-like apyrase remained in the insoluble fraction that was not used in the purification procedures. This work studied the construction of cDNA libraries with Hybrid Consensus-degenerate Oligonucleotide Primers (40bp) and low stringency RT-PCR. Introduction of touch-down PCR in the standard ORESTES technique improved the number of non-redundant sequences per library twofold. Association of touch-down with hybrid consensus-degenerated oligonucleotide primers increased this number to 300 sequences per library. The modifications resulted in a 10-fold decrease in the amount of work required for libraries construction and high-throughput sequencing (Fietto et al., 2000; addendum to patent application #196,716 - Ludwig Institute for Cancer Research). The best amplification profiles were obtained in RT-PCR reactions with low salt and a slow temperature ramp. Libraries made with shorter hybrid primers (25bp) showed more redundant sequences, reflecting a higher specificity for few templates. These shorter primers were used in combination with tegument mRNA to clone a gene fragment homologous to CD39 of mouse. This sequence was used in RACE-PCR reactions (rapid amplification of cDNA ends) to clone a 2.9 Kb cDNA corresponding to most of the predicted coding sequence of the Schistosoma gene.

O splicing alternativo é o processo pelo qual diversos transcritos podem ser gerados a partir de um único gene, sendo de extrema importância para diversidade do repertório transcricional e proteico. Diferentes variantes de splicing são expressas entre os diferentes tecidos e estágios de desenvolvimento garantindo o funcionamento normal da célula, portanto, qualquer alteração neste padrão pode resultar no aparecimento de doenças. Neste contexto, o objetivo deste trabalho foi o estabelecimento de metodologias para identificação de variantes de splicing em câncer de mama sob influência do oncogene ERBB2, o qual é um marcador de mau prognóstico altamente expresso em cerca de 30% dos tumores de mama. Foram estabelecidas duas estratégias para construção de bibliotecas de cDNA. A construção de bibliotecas de cDNA enriquecidas para splicing alternativo, baseada na formação e captura de moléculas de heteroduplexes em combinação com a amplificação de RNAm, foi realizada a partir de RNA total de linhagem celular de mama e a partir de um grupo de cinco amostras tumorais, todas com alta expressão de ERBB2. Foram identificadas 79 possíveis variantes de splicing alternativo em câncer de mama, das quais 18 foram selecionadas para validação por RT-PCR. Foi obtida uma taxa de vallidação de 94% e foram identificadas duas novas variantes de splicing alternativo. A regulação da expressão mediada por ERBB2 de três variantes de splicing foi confirmada por duas metodologias distintas, eletroforese em chip e estratégia baseada na ligação de sondas específicas, que revelou desbalanço de expressão entre as variantes, demonstrando a influência do oncogene na regulação de variantes de splicing. A segunda abordagem utilizada, foi a construção de bibliotecas de cDNA para avaliação do transcriptoma total, utilizando sequênciamento de alto desempenho. Foram utilizados RNA total de duas linhagens celulares de mama que diferem apenas na expressão do gene ERBB2. Foram identificadas 2.865 novas variantes de splicing, das quais 20, que reportaram a identificação de um novo éxon, foram selecionadas para validação, com uma taxa de validação de 90%. Seis destas variantes apresentaram aumento de expressão na linhagem com alta expressão de ERBB2. Além disso, foi detectado um enriquecimento de algumas categorias de variantes na linhagem celular com alta expressão de ERBB2, reforçando a influência do oncogene na regulação do splicing alternativo, podendo resultar em variantes de splicing associadas a este grupo de câncer de mama, que podem ser candidatas a marcadores moleculares. / Alternative splicing is a process, by which many differente transcripts can be generated by one single gene, significantly expanding the transcriptional and proteomic diversity. Different splicing variants are generated among different transcripts and developmental stages, assuring normal cell function. Therefore, alterations in the splicing process can lead to diseases outcome. In this context, the aim of this study was the establishment of methodologies for the identification of alternative splicing in breast cancer influenced by ERBB2 oncogene, which is a poor prognostic molecular marker, highly expressed in 30% of human breast cancer. Two strategies were established for the construction of cDNA libraries. Alternative enriched splicing libraries, based on heteroduplex capture combined with mRNA amplification, were constructed from total RNA from a cell line and also from five tumor samples, all of them presenting high ERBB2 expression. Seventy nine putative splicing variants were identified and 18 of them were selected for RT-PCR validation. A high validation level was obtained (94%) and two novel alternative splicing variants were identified. ERBB2 mediated regulation was confirmed for three variants by two distinct methodologies, electrophoresis on a chip and probe specific ligation approach. The alteration in the expression balance of variants suggests the influence of the oncogene in the splicing pattern regulation. The second strategy was the construction of cDNA libraries for global transcriptome analysis based on deep sequencing. Total RNA from two mammary epithelial cell lines expressing different ERBB2 levels were used and 2,865 novel splicing variants were identified. Twenty novel events reporting the inclusion of novel exons were selected for RT-PCR validation with 90% validation rate. Six variants presented higher expression in the cell line with high levels of ERBB2. Moreover enrichment in splicing events was detected in the ERBB2 high expressing cell line, supporting the ERBB2 influence in alternative splicing regulation, possibly resulting in splicing variants associated to this subgroup of cancer that can be tested as molecular markers.

Este trabalho mostrou a purificação de uma apirase em extratos de tegumento de vermes adultos de S. mansoni com razão de hidrólise de ADP/ATP aproximadamente 2. Análise da amostra por MALDI-TOF evidenciou a presença de helicase e carboxilesterase, sendo que a atividade de hidrólise de ADP ainda não foi descrita para estas proteínas. Através de \"Western blot\" mostrou-se que a proteína purificada não tem epítopos reconhecíveis por anticorpo anti-apirase de batata, e que a imunoreatividade cruzada com apirase da família CD39 está presente na fração insolúvel que não foi utilizada no processo de purificação. Esta tese caracteriza também a utilização de \"primers\" consenso degenerados na construção de bibliotecas de cDNA com PCR de baixa estringência. A introdução do \"touch-down\" PCR aumentou o número médio de sequências não redundantes por biblioteca de 20 para 40. O \"touch-down\" associado ao uso de primers híbridos consenso-degenerados (40 pb) elevou a média de sequências não redundantes para 300. Este número significa uma diminuição de cerca de 10 vezes na quantidade de trabalho de clonagem e sequenciamento necessário para se gerar o mesmo montante de dados (Fietto et al., 2000, addendum patent application Nº196,716). A diminuição da concentração de sal na reação e abaixamento da velocidade de rampa entre os passos de anelamento e extensão gerou os melhores perfis de amplificação de cDNA. Bibliotecas feitas com primers híbridos mais curtos (25 pb) mostraram-se mais redundantes, refletindo uma maior especificidade por certas sequências alvo. Com estes primers, usando-se RNAm de tegumento, clonamos um fragmento parcial de cDNA altamente homólogo à CD39 de camundongo. Esta sequência serviu como base para o isolamento de grande parte do gene de Schistosoma (2,9 Kb) através de RACE-PCR (Rapid Amplification of cDNA Ends). / This work shows purification of an apyrase from the tegument membrane of S. mansoni adult worms that exhibited an ADP/ATP hydrolysis ratio near 2. MALDI-TOF analyses showed that helicase and carboxylesterase were present in the sample, however in the literature there is no ADPase activity described for either enzyme. Western blot analyses showed that the purified apyrase ha no epitopes common to members of the CD39 apyrase family. On the other hand, a CD39-like apyrase remained in the insoluble fraction that was not used in the purification procedures. This work studied the construction of cDNA libraries with Hybrid Consensus-degenerate Oligonucleotide Primers (40bp) and low stringency RT-PCR. Introduction of touch-down PCR in the standard ORESTES technique improved the number of non-redundant sequences per library twofold. Association of touch-down with hybrid consensus-degenerated oligonucleotide primers increased this number to 300 sequences per library. The modifications resulted in a 10-fold decrease in the amount of work required for libraries construction and high-throughput sequencing (Fietto et al., 2000; addendum to patent application #196,716 - Ludwig Institute for Cancer Research). The best amplification profiles were obtained in RT-PCR reactions with low salt and a slow temperature ramp. Libraries made with shorter hybrid primers (25bp) showed more redundant sequences, reflecting a higher specificity for few templates. These shorter primers were used in combination with tegument mRNA to clone a gene fragment homologous to CD39 of mouse. This sequence was used in RACE-PCR reactions (rapid amplification of cDNA ends) to clone a 2.9 Kb cDNA corresponding to most of the predicted coding sequence of the Schistosoma gene.

A critical event during the life cycle of Dictyostelium discoideum is glycogen turnover. This process is catalyzed by glycogen phosphorylase-2 (gp-2). Since gp-2 expression is first induced during the transition from growth to differentiation, understanding how this gene is controlled may provide some insight into the process of differentiation. In order to identify the trans-acting factors responsible for activating gp-2 expression, cDNA plasmid libraries of amoebae, and cells at 8 h and 12 h of development were generated. The long-term goals of this project involve screening expression libraries with identified cis-acting elements from the gp-2 promoter to yield the DNA binding proteins responsible for gene regulation. For this approach to succeed, a high-quality cDNA library is essential. The library must contain full-length cDNA that represents the complexity of mRNA present during the developmental stage of interest. Hence, all three libraries were subjected to extensive testing prior to and following cloning. RNA quality and the fidelity of the time points were determined by Northern blot analysis and by RTPCR for several marker genes. Following cDNA synthesis, the cDNA was assessed for complexity and full-length synthesis by PCR and radioactive primer extension, respectively. Ligation of the cDNA into a vector was performed using several ratios of vector:insert in order to ensure that long cDNA species were included in the plasmid library. Finally, the presence of the marker genes was confirmed by PCR amplification of plasmid extracted from bacteria transformed with the plasmid library. / Master of Science

Thesis (MSc (Genetics))--Stellenbosch University, 2008. / The vine mealybug, Planococcus ficus (Signoret), is a severe pest of grapevine in many grape and wine producing countries around the world. It is renowned not only for the considerable damage it infers to grapevine of its own accord, but in particular for its role in transmitting deleterious viral diseases such as grapevine leafroll disease, Kober stem grooving, Shiraz disease and corky bark. Incidentally, it is an exceptionally tenacious antagonist of grapevine, being resistant to both chemical and biological control mechanisms. As a result, finding an effective strategy for P. ficus control has become a main priority of viticultural industries worldwide. Possible implementation of biotechnological approaches to pest management has resulted in a need for P. ficus genetic data - of which there are currently very little available. The transcribed genes of an organism can be captured in a cDNA library, and the sequences of the various transcripts can then be characterized. In this study altogether five cDNA libraries were constructed from the transcribed sequences of Planococcus ficus (Signoret). Instrumental to their construction was the identification of an RNA extraction protocol that provided large quantities of high quality RNA from mealybugs. The five cDNA libraries were the result of a set of modifications to the Creator™ SMART™ cDNA Library Construction Kit (used for Primary Library construction), and differed mainly with regards to range of insert sizes they contain. Whereas an abundance of short fragments were found in the Primary Library (42% of screened inserts 60.5 kb, and 20% >1 kb), the Fractionated Libraries contained inserts of specific size ranges that were more-or-less equally represented. The broadest size range was found in Fractionated Library 4, for which a uniform distribution over the range 0.25 kb - 4 kb was observed. Average insert sizes of Fractionated Libraries 1 to 4 were estimated at 0.25 kb, 0.5 kb, 1 kb and 2 kb respectively. These results demonstrated the importance of using a protocol designed to circumvent the bias towards incorporation of shorter transcripts in cDNA libraries. Although the libraries were not exhaustively analyzed, the outcome of a pilot investigation indicated that 41% of the submitted sequences had matches in the non-redundant database of the National Center for Biotechnology Information (NCBI, E-value 6 10-5), and that approximately 82% of these were of insect origin. Moreover, two potential targets for an RNAi-mediated approach to P. ficus pest control were identified. With one exception, these sequences seemed to be unique to arthropods. Future research needs to investigate the efficiency by which these sequences are able to constrain P. ficus proliferation, and their suitability for grapevine transformation.

Made available in DSpace on 2014-12-17T14:03:38Z (GMT). No. of bitstreams: 1 RenataKSE_DISSERT.pdf: 1992793 bytes, checksum: 77e111c6a7208973429d97e0cf1a51eb (MD5) Previous issue date: 2011-09-09 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Flowering is a fundamental process in the life cycle for plant. This process is marked by vegetative to reproductive apical meristem conversion, due to interactions between several factors, both internal and external to plant. Therefore, eight subtractive libraries were constructed using apical meristem induced or not induced for two contrasting species: Solanum lycopersicum cv. Micro-Tom and Solanum pimpinellifolium. Several cDNAs were identified and among these, were selected two cDNAs: one homologous cDNA to cyclophilin (LeCYP1) and the other to Auxin repressed protein (ARP). It has observed that LeCYP1 and ARP genes are important in the developmental process to plants. In silico analysis, were used several databases with the exclusion criterion E-value <1.0x10-15. As a result, conservation was observed for proteins analyzed by means of multiple alignments and the presence of functional domains. Then, overexpression cassettes were constructed for the ARP cDNA in sense and antisense orientations. For this step, it was used the CaMV35S promoter. The cDNA orientation (sense or antisense) in relation to the promoter was determined by restriction enzymes and sequencing. Then, this cassette was transferred to binary vector pZP211 and these cassettes were transferred into Agrobacterium tumefaciens LBA4404. S. lycopersicum cv. Micro-Tom (MT) and MT-Rg1 plants were transformed. In addition, seedlings were subjected to hormone treatments using a synthetic auxin (&#61537;- naphthalene acetic acid) and cyclosporin A (cyclophilin inhibitor) treatments and it was found that the hormone treatment there were changes in development of lateral roots pattern, probably related to decreases in auxin signaling caused by reduction of LeCYP1 in MT-dgt plants while cyclosporin A treatments, there was a slight delay in flowering in cv. MT plants. Furthermore, assay with real-time PCR (RT-qPCR) were done for expression level analysis from LeCYP1 and ARP in order to functionally characterize these sequences in tomato plants. / A flora??o ? fundamental no ciclo de vida de uma planta. Este processo ? marcado pela convers?o do meristema apical vegetativo em reprodutivo, devido a intera??es entre diversos fatores, tanto internos quanto externos ? planta. Diante disso, foram constru?das oito bibliotecas subtrativa utilizando ?pices meristem?ticos induzidos e n?o induzidos para a flora??o de duas esp?cies de tomateiro: Solanum lycopersicum cv Micro-Tom e S. pimpinellifolium. In?meros cDNAs foram identificados e dentre estes, foram selecionados o cDNA hom?logo a ciclofilina (LeCYP1) e a Prote?na Reprimida por Auxina (ARP) para a caracteriza??o in silico e funcional em esp?cie de tomateiro. Tem sido observado em outros sistemas vegetais que os genes LeCYP1 e ARP s?o importantes no processo de desenvolvimento da planta. Na an?lise in silico, foram utilizados diversos bancos de dados, tendo como crit?rio de exclus?o o E-value <1.0x10-15. Uma conserva??o para as prote?nas analisadas foi observada por meio dos alinhamentos m?ltiplos e a presen?a de dom?nios funcionais. Em seguida, foram realizadas constru??es de cassetes de superexpress?o para o cDNA hom?logo ? ARP em orienta??o senso e antissenso. Para esta etapa, foi utilizado o promotor forte CaMV35S presente no vetor intermedi?rio pBC (Stratagene). A orienta??o do cDNA (senso ou antissenso) em rela??o ao promotor foi determinada por meio de enzimas de restri??o e sequenciamento dos potenciais clones. Em seguida, este cassete foi transferido para o vetor bin?rio pZP211 e os clones obtidos foram confirmados pelo padr?o de restri??o utilizando diferentes enzimas. Subsequentemente, estes cassetes foram transferidos para a Agrobacterium tumefaciens LBA4404 e posteriormente transformado em plantas de S. lycopersicum cv. Micro-Tom (MT) e MT-Rg1. Em adi??o, pl?ntulas foram submetidas a tratamentos hormonais utilizando uma auxina sint?tica (?cido &#61537;-naftaleno ac?tico) e com ciclosporina A (inibidor de ciclofilina) e foi constatado que no tratamento hormonal houve altera??es no padr?o de desenvolvimento das ra?zes laterais, provavelmente relacionada ao d?ficit na sinaliza??o da auxina ocasionada pela redu??o de LeCYP1 em plantas MT-dgt, enquanto que no tratamento com ciclosporina A, ocorreu um atraso no florescimento de plantas cv MT. Al?m disso, ensaios de PCR em tempo real (RT-qPCR) foram efetuados para an?lise de express?o dos cDNAs hom?logos a LeCYP1 e ARP de modo a caracterizarmos funcionalmente estas sequ?ncias em plantas de tomateiro.