1,720,971 research outputs found
Evaluation of RNA Extraction Methods and Identification of Putative Reference Genes for Real-Time Quantitative Polymerase Chain Reaction Expression Studies on Olive (Olea europaeaL.) Fruits
No full textGenome wide transcriptomic surveys together with targeted molecular studies are uncovering an ever increasing number of differentially expressed genes in relation to agriculturally relevant processes in olive (Olea europaea L). These data need to be supported by quantitative approaches enabling the precise estimation of transcript abundance. qPCR being the most widely adopted technique for mRNA quantification, preliminary work needs to be done to set up robust methods for extraction of fully functional RNA and for the identification of the best reference genes to obtain reliable quantification of transcripts. In this work, we have assessed different methods for their suitability for RNA extraction from olive fruits and leaves and we have evaluated thirteen potential candidate reference genes on 21 RNA samples belonging to fruit developmental/ripening series and to leaves subjected to wounding. By using two different algorithms, GAPDH2 and PP2A1 were identified as the best reference genes for olive fruit development and ripening, and their effectiveness for normalization of expression of two ripening marker genes was demonstrated
Evaluation of candidate reference genes for qPCR in maize
No full textQuantitative real-time PCR (qPCR) is a powerful tool to measure gene expression levels. Accurate and
reproducible results are dependent on the correct choice of the reference genes for data normalization.
To date, screenings evaluating candidate reference gene stability for expression studies in maize have
not been reported. In the present work, we analyzed the expression patterns of 12 genes in a set of 20
maize samples, obtained from different tissues of plants grown at various experimental conditions. Using
genormPLUS, NormFinder and BestKeeper algorithms, the expression stability of three “classical” reference
genes, such as ACT, TUB and 18S rRNA, and the newly identified candidates, was assessed. With respect to
the algorithms, our results showed similar performance among genormPLUS, NormFinder and BestKeeper
in evaluating the suitability of reference genes. Our data therefore showed that the currently and widely
used reference genes for data normalization in maize were not the most stable expressed transcripts. Five
of the new putative reference genes (CUL, FPGS, LUG, MEP and UBCP) exhibited the highest expression
stability according to all algorithms. In conclusion, with this study, we provide a list of validated reference
genes and their relative primer sequences to conduct reliable qPCR experiments in maize
The miRNA-mediated post-transcriptional regulation of maize response to nitrate
No full textStress responses depend on the correct regulation of gene
expression. The discovery that abiotic as well as biotic stresses
can regulate miRNA levels, coupled with the identification
and functional analyses of stress-associated genes as miRNA
targets, provided clues about the vital role that several miRNAs
may play in modulating plant resistance to stresses. Nitrogen
availability seriously affects crops productivity and environment
and the understanding of the miRNA-guided stress
regulatory networks should provide new tools for the genetic
improvement of nitrogen use efficiency of crops. A recent
study revealed the potential role of a number of nitrateresponsive
miRNAs in the maize adaptation to nitrate fluctuations.
In particular, results obtained suggested that a nitrate
depletion might regulate the expression of genes involved
in the starvation adaptive response, by affecting the spatiotemporal
expression patterns of specific miRNAs
The dynamic regulation of microRNAs circuits in plant adaptation to abiotic stresses: A survey on molecular, physiological and methodological aspects
Full text linkBeing sessile organisms, plants continuously face a complex array of environmental stresses and have
naturally evolved multifaceted but well-coordinated adaptive responses to cope with them.
Recent studies in various plant species and varieties have established microRNAs (miRNAs) as key
elements in the post-transcriptional regulation of response to abiotic stresses. However, despite their
critical role has been widely recognized, the joint action of miRNAs with the main pathways of response
is still far from being completely clarified. Moreover, little is still known on the control of the biogenesis
and maturation of stress responsive miRNAs.
Here we give an overview on the involvement of microRNAs in plant response to nitrogen and phospho-
rous deprivation and on their less characterized role in the plant response to hypoxia and cold. Nitrogen
and phosphorous are the most limiting nutrients for agricultural production and the plant responsive-
ness to their deficiency is significantly regulated at the transcriptional and post-transcriptional level. We
reported the different miRNAs families involved, which sometimes are also common to different species
and other nutrient or abiotic stresses. Indeed we reported that some miRNAs families involved in N and
P deprivation are also acting in hypoxia and cold stresses. The results of the few existing studies on the
miRNAs involvement on low oxygen and cold conditions are also described.
Furthermore, we reviewed recent progress on methods for microRNAs localization/quantification and
de novo
identification, by exploring the main tools available and used
PRaTo: A web-tool to select optimal primer pairs for qPCR
No full textAn essential pre-requisite to perform sound quantitative real-time polymerase chain reaction (qPCR) assays is to design outstanding primer pairs. This means they must have a good efficiency and be not prone to produce multiple amplicons or primer dimer products. To circumvent these issues, several softwares are available to help primer design. Although satisfactory computer-aided primer design tools are available for standard PCR, less efforts were done to provide specific methods for selection of optimal primer pairs for qPCR. We have developed PRaTo a web-based tool that enables checking and ranking of primers pairs for their attitude to perform optimally and reliably when used in qPCR experiments. PRaTo is available at http://prato.daapv.unipd.it
Genome-wide discovery and characterization of nitrate-responsive miRNAs in roots of maize seedlings
No full textAbstract
Nitrogen availability affects crops productivity and environment. The natural abundance of useable nitrogen is so low that the massive human alteration of the nitrogen cycle has been required to sustain the feeding of the world's population. Tons of nitrogenous fertilizers are added to the soil worldwide annually, giving rise to environmental diseases. In this scenario, the knowledge of post-transcriptional regulation of plant response to nutrients is important to improve nitrogen use efficiency of crop. With the identification of stress-responsive miRNAs, a layer of post-transcriptional gene regulation has been uncovered.
We used a maize miRNAs-microarray platform to discover previously unknown nitrate-responsive miRNAs. Six mature miRNAs were identified and their expression profiles were studied by quantitative Real Time PCR (qPCR) and in situ hybridization (ISH) in maize roots grown in different nitrate availabilities.
Significant differences in miRNAs’ transcripts accumulation were evidenced between nitrate-supplied and nitrate-depleted roots. Real time PCR analyses and in situ detection of miRNAs confirmed the arrays data and evidenced distinct miRNAs spatiotemporal expression patterns in maize roots.
An in silico approach was used to select target genes of the miRNAs identified. Their transcripts accumulation has been investigated in both nitrate-supplied and nitrate-depleted roots by means of qPCR and ISH.
Our results suggest that miRNAs play some role in modulating N-responsive gene expression by inducing post-transcriptionally the expression of target genes. In particular, the repression of the transcription of miRNA identified upon nitrate shortage could represent a crucial step integrating nitrate signals into developmental changes in maize roots
Isolation and characterization of terpene synthases potentially involved in flavor development of ripening olive (Olea europaea) fruits
No full textThe flavor and taste of fruits are often determined by terpenes. We identified three cDNAs encoding putative terpene synthases from olive fruits of cv. Frantoio and Grignano. Heterologous expression in a bacterial system demonstrated that one of the terpene synthases, OeGES1, was an active monoterpene synthase that converted geranyl diphosphate to the monoterpene alcohol geraniol. The transcript accumulation pattern of this gene showed a peak during fruit ripening in both genotypes, indicating that the enzyme may be involved in the production of monoterpene flavor compounds in olive fruit. Although the putative terpene synthases OeTPS2 and OeTPS3 clustered with alpha-farnesene synthases and angiosperm monoterpene synthases, no detectable in vitro activity was found after expression in a bacterial system. Nevertheless, their transcripts sharply accumulated during fruit ripening starting from veraison
Differential expression and regulation of a neutral invertase encoding gene from peach (Prunus persica): evidence for a role in fruit development
No full textStudies on sucrose metabolism during fruit development have shown an
important role of acidic (cell wall and vacuolar) invertases (EC 3.2.1.26) in
determining fruit sink strength, final fruit growth and sugar accumulation. Little
information is available on the role played by neutral (cytoplasmic) invertases
on fruit and plant development. In this article, the expression of a gene
encoding a neutral invertase (NI) isolated from peach (PpNI1) was studied in
relation to sucrose metabolism and mesocarp development in two genotypes
(cv. Springcrest and cv. Redhaven) differing for fruit growth, and sugar
accumulation dynamics. Real-time reverse transcription-PCR expression
analyses showed a differential regulation of the gene during development
and a correlation with sucrose, and glucose and fructose mesocarp contents.
Only one peak of expression of the gene was found in the early ripening cultivar
Springcrest, characterised by a nearly monophasic growth of fruits, while two
peaks could be detected in the mid–late ripening cv. Redhaven, displaying
a classical biphasic double-sigmoidal fruit growth pattern. Furthermore, PpNI1
transcription appeared to be regulated in response to sugar signals only in the
phase of fruit expansion coincident with the onset of sucrose accumulation.
These findings point to a relationship between dynamics of fruit growth, sugar
metabolism and sensing and the expression of a gene encoding a NI, suggesting
a regulatory role in plant development for this class of enzymes
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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