RESUMO
Lead (Pb) is one of the most serious environmental pollutants. The aquatic fern Salvinia minima Baker is capable to hyper-accumulate Pb in their tissues. However, the molecular mechanisms involved in its Pb accumulation and tolerance capacity are not fully understood. In order to investigate the molecular mechanisms that are activated by S. minima in response to Pb, we constructed a suppression subtractive hybridization library (SSH) in response to an exposure to 40µM of Pb(NO3)2 for 12h. 365 lead-related differentially expressed sequences tags (ESTs) were isolated and sequenced. Among these ESTs, 143 unique cDNA (97 were registered at the GenBank and 46 ESTs were not registered, because they did not meet the GenBank conditions). Those ESTs were identified and classified into 3 groups according to Blast2GO. In terms of metabolic pathways, they were grouped into 29 KEGG pathways. Among the ESTs, we identified some that might be part of the mechanism that this fern may have to deal with this metal, including abiotic-stress-related transcription factors, some that might be involved in tolerance mechanisms such as ROS scavenging, membrane protection, and those of cell homeostasis recovery. To validate the SSH library, 4 genes were randomly selected from the library and analyzed by qRT-PCR. These 4 genes were transcriptionally up-regulated in response to lead in at least one of the two tested tissues (roots and leaves). The present library is one of the few genomics approaches to study the response to metal stress in an aquatic fern, representing novel molecular information and tools to understand the molecular physiology of its Pb tolerance and hyperaccumulation capacity. Further research is required to elucidate the functions of the lead-induced genes that remain classified as unknown, to perhaps reveal novel molecular mechanisms of Pb tolerance and accumulation capacity in aquatic plants.
Assuntos
Gleiquênias/efeitos dos fármacos , Chumbo/toxicidade , Nitratos/toxicidade , Poluentes Químicos da Água/toxicidade , Sequência de Bases , DNA Complementar/metabolismo , Etiquetas de Sequências Expressas , Gleiquênias/metabolismo , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Regulação para CimaRESUMO
Latex, the milky cytoplasm of highly differentiated cells called laticifers, from Hevea brasiliensis is a key source of commercial natural rubber production. One way to enhance natural rubber production would be to express genes involved in natural rubber biosynthesis by a laticifer-specific overexpression system. As a first step to identify promoters which could regulate the laticifer-specific expression, we identified random clones from a cDNA library of H. brasiliensis latex, resulting in 4325 expressed sequence tags (ESTs) assembled into 1308 unigenes (692 contigs and 617 singletons). Quantitative analyses of the transcription levels of high redundancy clones in the ESTs revealed genes highly and predominantly expressed in laticifers, such as Rubber Elongation Factor (REF), Small Rubber Particle Protein and putative protease inhibitor proteins. HRT1 and HRT2, cis-prenyltransferases involved in rubber biosynthesis, was also expressed predominantly in laticifers, although these transcript levels were 80-fold lower than that of REF. The 5'-upstream regions of these laticifer-specific genes were cloned and analyzed in silico, revealing seven common motifs consisting of eight bases. Furthermore, transcription factors specifically expressed in laticifers were also identified. The common motifs in the laticifer-specific genes and the laticifer-specific transcription factors are potentially involved in the regulation of gene expression in laticifers.
Assuntos
Regulação da Expressão Gênica de Plantas , Genes de Plantas , Hevea/genética , Látex/biossíntese , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Borracha , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar , Dimetilaliltranstransferase/metabolismo , Etiquetas de Sequências Expressas , Expressão Gênica , Biblioteca Gênica , Hevea/metabolismo , Dados de Sequência Molecular , Células Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In several plant tissues, programmed cell death (PCD) is mediated by the combined action of cysteine peptidases, namely KDEL-tailed cysteine peptidases (KDEL-CysEP) and vacuolar processing enzymes (VPE). Here, we performed a search of the draft genome of Jatropha curcas L. (Euphorbiaceae) and identified 2 genes for KDEL-CysEP (Jc-CysEP1 and Jc-CysEP2) and 3 genes for VPE (Jc-ßVPE, Jc-γVPE and Jc-δVPE) and determined the expression patterns of these genes by RT-qPCR in integument and cellular endosperm of seeds collected at seven different developmental stages. We were able to demonstrate that the expression of Jc-CysEP1, Jc-CysEP2, Jc-ßVPE and Jc-γVPE proceeded rapidly from Stage IV, with Jc-CysEP2 displaying the highest relative expression; expression of Jc-δVPE could not be detected in any of the tissues/developmental stages analyzed. Additionally, we showed that the expression pattern of these peptidases correlates with anatomical changes in integument and cellular endosperm, thus suggesting a role for both classes of peptidases in PCD and in protein processing, both of which occur simultaneously in each of these tissues.
Assuntos
Cisteína Endopeptidases/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Jatropha/genética , Apoptose , Cisteína/metabolismo , Cisteína Endopeptidases/metabolismo , Primers do DNA/genética , Endosperma/citologia , Endosperma/genética , Endosperma/crescimento & desenvolvimento , Endosperma/fisiologia , Genômica , Jatropha/citologia , Jatropha/crescimento & desenvolvimento , Jatropha/fisiologia , Oligopeptídeos , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sinais Direcionadores de Proteínas , Sementes/citologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/fisiologiaRESUMO
La identificación de genes nuevos relacionados con la respuesta de Nicotiana tabacum L. al estrés biótico y abiótico contribuye al mejoramiento genético del cultivo del tabaco en todo el mundo. El objetivo de este trabajo fue detectar la presencia de los genes tpt1 y pr1 en el genoma de algunas especies y variedades cubanas de tabaco. Se identificaron 265 etiquetas de secuencias expresadas (ESTs-expressed sequences tags) que nunca se habían informado con anterioridad en especies vegetales, y el gen que codifica para la proteína tumoral controlada durante la transcripción (Transcriptional Controlled Tumor Protein) (tpt1) nunca se había informado en N. tabacum, pues los estudios del mismo se han centrado en su mayoría en animales y humanos. Los resultados del microarreglo se confirmaron utilizando la RT-PCR cuantitativa en tiempo real. Los genes tpt1 y proteína relacionada con la patogénesis (pr1) se utilizaron para diseñar cebadores para la caracterización molecular de algunas especies y variedades de tabaco cubano. El gen tpt1 solamente se expresó en dos especies (N. glutinosa y N. tomentosiformis) y el pr1, después de la digestión, mostró diferentes bandas entre las variedades susceptibles y resistentes. La presencia de estos genes en una parte del germoplasma analizado constituye un paso inicial para la utilización de este conocimiento en el mejoramiento genético del tabaco.
The identification of new genes related with Nicotiana tabacum L. response to biotic and abiotic stress contribute to the genetic improvement of tobacco plants around the world. The aim of this research was to identify tpt1 and pr1 genes in the genomic of some cuban tobacco varieties and species. The microarray technology has been used with ESTs for the construction of a cDNA library. 265 expressed sequences tags (ESTs) that have never been reported before in vegetables species were identified and the gene that codified for the Transcriptional Controlled Tumor Protein (tpt1), has never been reported before in N. tabacum, with the studies about this gene being focused on human and animals mostly. The microarray results were confirmed using quantitative RT- PCR. TCTP and pathogenesis-related protein 1 (PR-1) ESTs were used to design primers for the molecular characterization of some species and cuban tobacco varieties. The tpt1 gene was only expressed in two species (N. glutinosa y N. tomentosiformis) and pr1 gen, after a digestion, exhibited different bands for susceptible and resistant varieties. The detection of these genes in some species and cuban tobacco varieties represents one step to go deeply in the molecular characterization of cuban germoplasm.
Assuntos
Genes , Modelos Moleculares , Tabacum , Nicotiana , Estrutura MolecularRESUMO
Little is known about the evolutionary relationships among Linum species, basically because of the lack of transferable molecular markers. Currently, expressed sequence tags available in public databases provide an opportunity for the rapid and inexpensive development of simple sequence repeat (SSR) markers in wild flax species. In this regard, fifty expressed sequence tag-derived microsatellite markers (EST-SSRs) were evaluated for polymorphism and transferability in 50 Linum usitatissimum cultivars/accessions and 11 Linum species. Among them 23 EST-SSRs were polymorphic in L. usitatissimum, while 2-4 alleles were detected (average 2.26 per locus). The polymorphism information content value ranged from 0.08 to 0.55 (average 0.38). Forty one genic markers (95.3 percent) produced strong amplicons in at least two of the 11 Linum species. The percentage of cross amplification ranged from 34.1 percent to 92.7 percent in L. tauricum and L. bienne, respectively. Moreover, the rate of transferability was associated positively with the botanical section. Our results suggest that the high degree of EST-SSRs transferability to Linum species can be a useful enhancement of the current database of SSR markers for future genetic and evolutionary studies.
Assuntos
Etiquetas de Sequências Expressas , Linho/genética , Repetições de Microssatélites , Eletroforese , Linho , Reação em Cadeia da Polimerase , Polimorfismo Genético , Sementes/genéticaRESUMO
Pleiotropic drug resistance (PDR) proteins, a subfamily of the ATP-binding cassette (ABC) transporters, have been recently shown to play a role in plant defense against biotic and abiotic stresses. However, nothing is known about their expression in citrus. To investigate the occurrence of PDR homologues in citrus species, we have surveyed EST sequences from different tissues and conditions of the Citrus Expressed Sequence Tags (CitEST) database, through sequence similarity search analyses and inspections for characteristic PDR domains. Multiple sequence alignments, prediction of transmembrane topology and phylogenetic analysis of PDR-like proteins were additionally performed. This study allowed the identification of nine putative proteins showing characteristic PDR features in citrus species under various conditions, which may indicate a potential correlation between PDRs and stress and metabolism of citrus plants. Moreover, a tissue-specific putative PDR-like protein was found in sweet orange fruits. To our knowledge, this is the first report regarding the identification of citrus ESTs encoding PDR-like proteins as well as the first to identify a putative full ABC transporter with specific expression in fruits.
RESUMO
Slippage is an important sequencing problem that can occur in EST projects. However, very few studies have addressed this. We propose three new methods to detect slippage artifacts: arithmetic mean method, geometric mean method, and echo coverage method. Each method is simple and has two different strategies for processing sequences: suffix and subsequence. Using the 291,689 EST sequences produced in the SUCEST project, we performed comparative tests between our proposed methods and the SUCEST method. The subsequence strategy is better than the suffix strategy, because it is not anchored at the end of the sequence, so it is more flexible to find slippage at the beginning of the EST. In a comparison with the SUCEST method, the advantage of our methods is that they do not discard the majority of the sequences marked as slippage, but instead only remove the slipped artifact from the sequence. Based on our tests the echo coverage method with subsequence strategy shows the best compromise between slippage detection and ease of calibration.
Assuntos
Humanos , Análise de Sequência de DNA/métodos , Etiquetas de Sequências Expressas , Modelos Genéticos , Saccharum/genética , Técnicas Genéticas , Rearranjo Gênico , Replicação do DNARESUMO
The RNA biogenesis machinery of Paracoccidioides brasiliensis was assessed by comparative analyses of PbAESTs (P. brasiliensis assembled expressed sequence tags (ESTs)) with sequences from Saccharomyces cerevisiae MIPS database. PbAESTs related to almost all categories of S. cerevisiae RNA biogenesis were found. Two of the 12 S. cerevisiae RNA Pol II core subunits, Rpb3 and Rpb7, were found, probably reflecting the growth phase from which the cDNA libraries used in ESTs generation were constructed, as well as the low abundance of some of these transcripts. We have also found orthologs to TATA-box-binding protein (TBP), and at least one subunit of each TBP-associated factors (TFII) in P. brasiliensis transcriptome, except TFIIB. Genes associated to the chromatin remodeling complex, as well as transcription factors probably involved in the control of genes associated to a sexual cycle and virulence, were also identified. With respect to the pre-mRNA processing, 65 PbAEST orthologs to S. cerevisiae basal splicing machinery and 21 orthologs of 5'- and 3'-end formation processes were found. Components involved in RNA interference were detected, suggesting that this gene expression regulation mechanism is probably used by P. brasiliensis. Twelve PbAESTs related to Pol I and Pol III machineries were assigned as S. cerevisiae orthologs. Finally, 25 and 10 PbAESTs associated to rRNA and tRNA processing, respectively, were detected. Taken together, our results enable us to depict, for the first time, a global view of transcription and RNA processing in P. brasiliensis.
Assuntos
Humanos , Origem da Vida , Etiquetas de Sequências Expressas , Fatores de Transcrição/genética , Paracoccidioides/genética , Fatores de Transcrição/fisiologia , Genoma Fúngico , Paracoccidioides/fisiologia , RNA Fúngico/genética , RNA Polimerase II/genética , RNA Polimerase II/fisiologia , Reprodução , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Transcrição Gênica/fisiologiaRESUMO
A survey of the transcriptional profile of Haementeria depressa Ringuelet, 1972 (Annelida, Hirudinea) salivary complexes was produced through expressed sequence tag (EST). Sequences from 898 independent clones were assembled in 555 clusters, representing the transcript profile of this tissue. The repertoire of possible proteins involved in feeding and host interaction processes of the leech corresponded to 10.6% of all identified transcripts (67 clusters), being the carbonic anhydrases (30%), several coagulation inhibitors (25%) and hemerythrin-like molecules (19%), the major components. Among the 387 clusters matching cellular proteins, the majority represents molecules involved in gene and protein expression, reflecting a high specialization of this tissue for protein synthesis. Our H. depressa dbEST was also compared to those from other blood-feeding organisms, providing evidences that among the secreted proteins, the coagulation inhibitors present a profile very characteristic of this animal class