Nitrogen assimilation in Citrus based on CitEST data mining

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

N-glycosylation in sugarcane

The N-linked glycosylation of secretory and membrane proteins is the most complex posttranslational modification known to occur in eukaryotic cells. It has been shown to play critical roles in modulating protein function. Although this important biological process has been extensively studied in mammals, much less is known about this biosynthetic pathway in plants. The enzymes involved in plant N-glycan biosynthesis and processing are still not well defined and the mechanism of their genetic regulation is almost completely unknown. In this paper we describe our first attempt to understand the N-linked glycosylation mechanism in a plant species by using the data generated by the Sugarcane Expressed Sequence Tag (SUCEST) project. The SUCEST database was mined for sugarcane gene products potentially involved in the N-glycosylation pathway. This approach has led to the identification and functional assignment of 90 expressed sequence tag (EST) clusters sharing significant sequence similarity with the enzymes involved in N-glycan biosynthesis and processing. The ESTs identified were also analyzed to establish their relative abundance.

A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs

In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5′ sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.

Putative pyrophosphate phosphofructose 1-kinase genes identified in sugarcane may be getting energy from pyrophosphate

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) has been detected in several types of plant cells, but the gene has not been reported in sugar cane. Using Citrux paradixi PPi-PFK gene (AF095520 and AF095521) sequences to search the sugar cane EST database, we have identified both the α and β subunits of this enzyme. The deduced amino acid sequences showed 76 and 80% similarity with the corresponding α and β subunits of C. paradisi. A high degree of similarity was also observed among the PFK β subunits when the alignment of the sugar cane sequences was compared to those of Ricinus communis and Solanum tuberosum, it appears that α and β are two distinct subunits; they were found at different concentrations in several sugar cane tissues. It remains to be determined if the different gene expression levels have some physiological importance and how they affect sucrose synthesis, export, and storage in vacuoles. A comparison between the amino acid sequences of β PFKs from a variety of organisms allowed us to identify the two critical Asp residues typical of this enzyme's activity site and the other binding sites; these residues are tightly conserved in all members of this protein family. Apparently, there are catalytic residues on the β subunit of the pyrophosphate-dependent enzyme.

The post-transcriptional gene silencing pathway in Eucalyptus

Post-transcriptional gene silencing (PTGS) is a conserved surveillance mechanism that identifies and cleaves double-stranded RNA molecules and their cellular cognate transcripts. The RNA silencing response is actually used as a powerful technique (named RNA interference) for potent and specific inhibition of gene expression in several organisms. To identify gene products in Eucalyptus sharing similarities with enzymes involved in the PTGS pathway, we queried the expressed sequence tag database of the Brazilian Eucalyptus Genome Sequence Project Consortium (FORESTs) with the amino acid sequences of known PTGS-related proteins. Among twenty-six prospected genes, our search detected fifteen assembled sequences encoding products presenting high level of similarity (E value < 10 -40) to proteins involved in PTGS in plants and other organisms. We conclude that most of the genes known to be involved in the PTGS pathway are represented in the FORESTs database. Copyright by the Brazilian Society of Genetics.

Eucalyptus ESTs corresponding to the protoporphyrinogen IX oxidase enzyme related to the synthesis of heme, chlorophyll, and to the action of herbicides

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Eucalyptus ESTs corresponding to the enzyme glutamine synthetase and the protein D1, sites of action of herbicides that cause oxidative stress

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Eucalyptus ESTs associated with resistance to herbicide inhibitors of aromatic and branched-chain amino acid synthesis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification and frequency of transposable elements in Eucalyptus

Transposable elements (TE) are major components of eukaryotic genomes and involved in cell regulation and organism evolution. We have analyzed 123,889 expressed sequence tags of the Eucalyptus Genome Project database and found 124 sequences representing 76 TE in 9 groups, of which copia, MuDR and FAR1 groups were the most abundant. The low amount of sequences of TE may reflect the high efficiency of repression of these elements, a process that is called TE silencing. Frequency of groups of TE in Eucalyptus libraries which were prepared with different tissues or physiologic conditions from seedlings or adult plants indicated that developing plants experience the expression of a much wider spectrum of TE groups than that seen in adult plants. These are preliminary results that identify the most relevant TE groups involved with Eucalyptus development, which is important for industrial wood production. Copyright by the Brazilian Society of Genetics.

Eucalyptus ESTs involved in the production of 9-cis epoxycarotenoid dioxygenase, a regulatory enzyme of abscisic acid production

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Spatio-temporal distribution of fatty acid-binding protein 6 (fabp6) gene transcripts in the developing and adult zebrafish (Danio rerio)

We have determined the structure of the fatty acid-binding protein 6 (fabp6) gene and the tissue-specific distribution of its transcripts in embryos, larvae and adult zebrafish (Danio rerio). Like most members of the vertebrate FABP multigene family, the zebrafish fabp6 gene contains four exons separated by three introns. The coding region of the gene and expressed sequence tags code for a polypeptide of 131 amino acids (14 kDa, pI 6.59). The putative zebrafish Fabp6 protein shared greatest sequence identity with human FABP6 (55.3%) compared to other orthologous mammalian FABPs and paralogous zebrafish Fabps. Phylogenetic analysis showed that the zebrafish Fabp6 formed a distinct clade with the mammalian FABP6s. The zebrafish fabp6 gene was assigned to linkage group (chromosome) 21 by radiation hybrid mapping. Conserved gene synteny was evident between the zebrafish fabp6 gene on chromosome 21 and the FABP6/Fabp6 genes on human chromosome 5, rat chromosome 10 and mouse chromosome 11. Zebrafish fabp6 transcripts were first detected in the distal region of the intestine of embryos at 72 h postfertilization. This spatial distribution remained constant to 7-day-old larvae, the last stage assayed during larval development. In adult zebrafish, fabp6 transcripts were detected by RT-PCR in RNA extracted from liver, heart, intestine, ovary and kidney (most likely adrenal tissue), but not in RNA from skin, brain, gill, eye or muscle. In situ hybridization of a fabp6 riboprobe to adult zebrafish sections revealed intense hybridization signals in the adrenal homolog of the kidney and the distal region of the intestine, and to a lesser extent in ovary and liver, a transcript distribution that is similar, but not identical, to that seen for the mammalian FABP6/Fabp6 gene. © 2008 The Authors.

Screening of genomic libraries

Microsatellites, or simple sequence repeats (SSRs), have proven to be an important molecular marker in plant genetics and breeding research. The main strategies to obtain these markers can be through genomic DNA and from expressed sequence tags (ESTs) from mRNA/cDNA libraries. Genetic studies using microsatellite markers have increased rapidly because they can be highly polymorphic, codominant markers and they show heterozygous conserved sequences. Here, we describe a methodology to obtain microsatellite using the enrichment library of DNA genomic sequences. This method is highly efficient to development microsatellite markers especially in plants that do not have available ESTs or genome databases. This methodology has been used to enrich SSR marker libraries in Citrus spp., an important tool to genotype germplasm, to select zygotic hybrids, and to saturate genetic maps in breeding programs. © Springer Science+Business Media, LLC 2013.

Caracterização de uma região genômica relacionada a uma anomalia de viveiro em Eucalyptus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Expressão gênica diferencial em palmitos de cana-de-açúcar submetida a diferentes períodos de estresse hídrico

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Expression Pattern and Promoter Analysis of a Eucalyptus grandis Germin-like Gene

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)