Expression profiling of functional genes in prenatal skeletal muscle tissue in Duroc and Pietrain pigs
Davoli, R. ; Braglia, S. ; Russo, V. ; Varona, L. ; Pas, M.F.W. te - \ 2011
Journal of Animal Breeding and Genetics 128 (2011)1. - ISSN 0931-2668 - p. 15 - 27.
time rt-pcr - cyclin g1 - porcine fetal - sequence tags - family - protein - growth - cells - differentiation - cyclophilin
In livestock, skeletal muscle is a tissue of major economic importance for meat production and muscle mass is largely determined during the prenatal period by the number and the size of muscle fibres. The understanding of gene expression changes during prenatal pig muscle development is still limited. In this study, genes identified as differentially expressed in a previous microarray research and chosen for the function of the coded protein as putative candidate involved in myogenesis were considered to analyse their expression profile during foetal growth of Duroc and Pietrain pigs. The eleven genes were considered by real-time PCR for a time-course evaluation of the transcription level at six stages of prenatal longissimus dorsi development. The results suggest that the most relevant variations in mRNA levels of the analysed genes seem to follow temporal waves of gene expression. Significant changes of transcription were observed at 21–35 and 63–91 days, the two main phases of skeletal muscle development. During the early phases of Pietrain embryos’ development, 10 of the 11 genes showed an induction. In Duroc embryos, a second phase of gene up-regulation can be identified in the phase 63–77 days. These results provide new data on developmental changes of expression profile of 11 genes involved in different functional pathways related to prenatal myogenic processes in Duroc and Pietrain pigs.
Use of homologous and heterologous gene expression profiling tools to characterize transcription dynamics during apple fruit maturation and ripening
Costa, F. ; Alba, R. ; Schouten, H.J. ; Soglio, V. ; Gianfranceschi, L. ; Serra, S. ; Musacchi, S. ; Sansavini, S. ; Costa, G. ; Fei, Z. ; Giovannoni, J. - \ 2010
BMC Plant Biology 10 (2010). - ISSN 1471-2229 - 36 p.
cdna microarrays - ethylene biosynthesis - climacteric fruit - sequence tags - melon fruit - tomato - genomics - identification - morphogenesis - biochemistry
Background - Fruit development, maturation and ripening consists of a complex series of biochemical and physiological changes that in climacteric fruits, including apple and tomato, are coordinated by the gaseous hormone ethylene. These changes lead to final fruit quality and understanding of the functional machinery underlying these processes is of both biological and practical importance. To date many reports have been made on the analysis of gene expression in apple. In this study we focused our investigation on the role of ethylene during apple maturation, specifically comparing transcriptomics of normal ripening with changes resulting from application of the hormone receptor competitor 1-Methylcyclopropene. Results - To gain insight into the molecular process regulating ripening in apple, and to compare to tomato (model species for ripening studies), we utilized both homologous and heterologous (tomato) microarray to profile transcriptome dynamics of genes involved in fruit development and ripening, emphasizing those which are ethylene regulated. The use of both types of microarrays facilitated transcriptome comparison between apple and tomato (for the later using data previously published and available at the TED: tomato expression database) and highlighted genes conserved during ripening of both species, which in turn represent a foundation for further comparative genomic studies. The cross-species analysis had the secondary aim of examining the efficiency of heterologous (specifically tomato) microarray hybridization for candidate gene identification as related to the ripening process. The resulting transcriptomics data revealed coordinated gene expression during fruit ripening of a subset of ripening-related and ethylene responsive genes, further facilitating the analysis of ethylene response during fruit maturation and ripening. Conclusion - Our combined strategy based on microarray hybridization enabled transcriptome characterization during normal climacteric apple ripening, as well as definition of ethylene-dependent transcriptome changes. Comparison with tomato fruit maturation and ethylene responsive transcriptome activity facilitated identification of putative conserved orthologous ripening-related genes, which serve as an initial set of candidates for assessing conservation of gene activity across genomes of fruit bearing plant species
Flexible tools for gene expression and silencing in tomato
Fernandez, A.I. ; Viron, N. ; Alhagdow, M. ; Karimi, M. ; Jones, M. ; Amsellem, Z. ; Sicard, A. ; Czerednik, A. ; Angenent, G.C. ; Grierson, D. ; May, S. ; Seymour, G. ; Eshed, Y. ; Lemaire-Chamley, M. ; Rothan, C. ; Hilson, P. - \ 2009
Plant Physiology 151 (2009)4. - ISSN 0032-0889 - p. 1729 - 1740.
zinc-finger - artificial micrornas - functional-analysis - sequence tags - crabs-claw - fruit - arabidopsis - plants - polygalacturonase - transcription
As a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collections and ease of cultivation and transformation that enable the analysis of biological processes impossible to investigate in other model species. To facilitate the assembly of an open genetic toolbox designed to study Solanaceae, we initiated a joint collection of publicly available gene manipulation tools. We focused on the characterization of promoters expressed at defined time windows during fruit development, for the regulated expression or silencing of genes of interest. Five promoter sequences were captured as entry clones compatible with the versatile MultiSite Gateway format: PPC2, PG, TPRP, and IMA from tomato and CRC from Arabidopsis (Arabidopsis thaliana). Corresponding transcriptional fusions were made with the GUS gene, a nuclear-localized GUS-GFP reporter, and the chimeric LhG4 transcription factor. The activity of the promoters during fruit development and in fruit tissues was confirmed in transgenic tomato lines. Novel Gateway destination vectors were generated for the transcription of artificial microRNA (amiRNA) precursors and hairpin RNAs under the control of these promoters, with schemes only involving Gateway BP and LR Clonase reactions. Efficient silencing of the endogenous phytoene desaturase gene was demonstrated in transgenic tomato lines producing a matching amiRNA under the cauliflower mosaic virus 35S or PPC2 promoter. Lastly, taking advantage of the pOP/LhG4 two-component system, we found that well-characterized flower-specific Arabidopsis promoters drive the expression of reporters in patterns generally compatible with heterologous expression. Tomato lines and plasmids will be distributed through a new Nottingham Arabidopsis Stock Centre service unit dedicated to Solanaceae resources
Differential expression of genes involved in C1 metabolism and lignin biosynthesis in wooden core and bast tissues of fibre hemp (Cannabis sativa L.).
Broeck, H.C. van den; Maliepaard, C.A. ; Ebskamp, M.J.M. ; Toonen, M.A.J. ; Koops, A.J. - \ 2008
Plant Science 174 (2008)2. - ISSN 0168-9452 - p. 205 - 220.
aspen populus-tremuloides - cellulose synthase gene - cell-wall biosynthesis - loblolly-pine - functional genomics - usitatissimum l. - sequence tags - xylem - arabidopsis - lignification
Plants are the major source of fibres for, e.g., textile and paper applications. Fibre hemp (Cannabis sativa L.) can be grown under a wide variety of agro-ecological conditions, is resistant to weeds and pests and, in general, drought tolerant. Fibre length and content of cellulose and lignin are important quality parameters for raw material used in cordage, textile, paper, and fibreboard industries. To improve currently used hemp varieties, more knowledge about the molecular processes that underlie cell wall metabolism is needed. To study gene expression in hemp, separate cDNA libraries were constructed from developing core fibres and developing and near mature bast fibres. A cDNA microarray was constructed with 3414 randomly selected hemp cDNAs. From these a total number of 1571 sequences was obtained from the 50 ends of the cDNAs. After similarity search, 1036 unique contigs were obtained. To study differences in the genetic background of quality properties of bast and core fibres, bast and core tissues were obtained from middle parts of the stems during various developmental stages and expression of the cDNAs was analyzed. A total number of 178 clones encoding 65 proteins was found to be more highly expressed in core tissue. Most of these 65 proteins are involved in C1 metabolism and lignin biosynthesis. In bast tissue, a total number of 110 clones encoding 44 proteins was more highly expressed. Among these 44 proteins are arabinogalactan proteins, lipid transfer proteins, lipoxygenases, and endoxyloglucan transferases. This paper describes the identification of genes that are differentially expressed in bast fibres and wooden core tissue of hemp, and is the first step in linking the genetic background to quality differences between these two types of hemp tissue. This research may result in biotechnological approaches to increase the value of hemp grown for industrial use
Genome-wide screening for cis-regulatory variation using a classical diallel crossing scheme
Kiekens, R. ; Vercauteren, A. ; Moerkerke, B. ; Goetghebeur, E. ; Daele, H. Van Den; Sterken, R. ; Kuiper, M. ; Eeuwijk, F.A. van; Vuylsteke, M. - \ 2006
Nucleic acids research 34 (2006)13. - ISSN 0305-1048 - p. 3677 - 3686.
human gene-expression - allelic variation - transcriptional regulation - arabidopsis-thaliana - functional genomics - sequence tags - microarray - model - identification - construction
Large-scale screening studies carried out to date for genetic variants that affect gene regulation are generally limited to descriptions of differences in allele-specific expression (ASE) detected in vivo. Allele-specific differences in gene expression provide evidence for a model whereby cis-acting genetic variation results in differential expression between alleles. Such gene surveys for regulatory variation are a first step in identifying the specific nucleotide changes that govern gene expression differences, but they leave the underlying mechanisms unexplored. Here, we propose a quantitative genetics approach to perform a genome-wide analysis of ASE differences (GASED). The GASED approach is based on a diallel design that is often used in plant breeding programs to estimate general combining abilities (GCA) of specific inbred lines and to identify high-yielding hybrid combinations of parents based on their specific combining abilities (SCAs). In a context of gene expression, the values of GCA and SCA parameters allow cis- and trans-regulatory changes to be distinguished and imbalances in gene expression to be ascribed to cis-regulatory variation. With this approach, a total of 715 genes could be identified that are likely to carry allelic polymorphisms responsible for at least a 1.5-fold allelic expression difference in a total of 10 diploid Arabidopsis thaliana hybrids. The major strength of the GASED approach, compared to other ASE detection methods, is that it is not restricted to genes with allelic transcript variants. Although a false-positive rate of 9/41 was observed, the GASED approach is a valuable pre-screening method that can accelerate systematic surveys of naturally occurring cis-regulatory variation among inbred lines for laboratory species, such as Arabidopsis, mouse, rat and fruitfly, and economically important crop species, such as corn.
Comparison of prenatal musle tissue expression profiles of two pigs breed differing in muscle characteristics
Cagnazzo, M. ; Pas, M.F.W. te; Priem, J. ; Wit, A.A.C. de; Pool, M.H. ; Davoli, R. ; Russo, V. - \ 2006
Journal of Animal Science 84 (2006). - ISSN 0021-8812 - p. 1 - 10.
skeletal-muscle - sequence tags - myogenesis - growth - gene - differentiation - selection - family
The objective of this study was to compare purebred Duroc and Pietrain prenatal muscle tissue transcriptome expression levels at different stages of prenatal development to gain insight into the differences in muscle tissue development in these pig breeds. Commercial western pig breeds have been selected for muscle growth for the past 2 decades. Pig breeds differ for their muscle phenotypes (i.e., myofiber numbers and myofiber types). Duroc and Pietrain pig breeds are extremes; Duroc pigs have redder muscle fiber types with more intramuscular fat, and Pietrain pigs have faster-growing and whiter muscle fiber types. Pietrain pigs are more muscular than Duroc pigs, whereas Duroc pigs are fatter than Pietrain pigs. The genomic background underlying these breed-specific differences is poorly known. Myogenesis is a complex exclusive prenatal process involving proliferation and differentiation (i.e., fusion) of precursor cells called myoblasts. We investigated the difference in the prenatal muscle-specific transcriptome profiles of Duroc and Pietrain pigs using microarray technology. The microarray contained more than 500 genes affecting myogenesis, energy metabolism, muscle structural genes, and other genes from a porcine muscle cDNA library. The results indicated that the expression of the myogenesis-related genes was greater in early Duroc embryos than in early Pietrain embryos (14 to 49 d of gestation), whereas the opposite was found in late embryos (63 to 91 d of gestation). These findings suggest that the myogenesis process is more intense in early Duroc embryos than in Pietrain embryos but that myogenesis is more intense in late Pietrain fetuses than in Duroc fetuses. Transcriptomes of muscle structural genes followed that pattern. The energy metabolism genes were expressed at a higher level in prenatal Pietrain pigs than in prenatal Duroc pigs, except for d 35, when the opposite situation was found. Fatty acid metabolism genes were expressed at a higher level in early (14 to 49 d of gestation) Duroc embryos than in Pietrain embryos. Better understanding of the genomic regulation of tissue formation leads to improved knowledge of the genome under selection and may lead to directed breed-specific changes in the future.