Quantitative RT-PCR based platform for rapid quantification of the transcripts of highly homologous multigene families and their members during grain development

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Quantitative RT-PCR based platform for rapid quantification of the transcripts of highly homologous multigene families and their members during grain development. / Kaczmarczyk, Agnieszka Ewa; Bowra, Steve; Elek, Zoltan; Vincze, Éva.

In: B M C Plant Biology, Vol. 12, No. 184, 2012.

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@article{b847ec3c6c8a4902b2829d390f278743,
title = "Quantitative RT-PCR based platform for rapid quantification of the transcripts of highly homologous multigene families and their members during grain development",
abstract = "BackgroundCereal storage proteins represent one of the most important sources of protein for food and feed and they are coded by multigene families. The expression of the storage protein genes exhibits a temporal fluctuation but also a response to environmental stimuli. Analysis of temporal gene expression combined with genetic variation in large multigene families with high homology among the alleles is very challenging.ResultsWe designed a rapid qRT-PCR system with the aim of characterising the variation in the expression of hordein genes families. All the known D-, C-, B-, and gamma-hordein sequences coding full length open reading frames were collected from commonly available databases. Phylogenetic analysis was performed and the members of the different hordein families were classified into subfamilies. Primer sets were designed to discriminate the gene expression level of whole families, subfamilies or individual members. The specificity of the primer sets was validated before successfully applying them to a cDNA population derived from developing grains of field grown Hordeum vulgare cv. Barke. The results quantify the number of moles of transcript contributed to a particular gene family and its subgroups. More over the results indicate the genotypic specific gene expression.ConclusionsQuantitative RT-PCR with SYBR Green labelling can be a useful technique to follow gene expression levels of large gene families with highly homologues members. We showed variation in the temporal expression of genes coding for barley storage proteins. The results imply that our rapid qRT-PCR system was sensitive enough to identify the presence of alleles and their expression profiles. It can be used to check the temporal fluctuations in hordein expressions or to find differences in their response to environmental stimuli. The method could be extended for cultivar recognition as some of the sequences from the database originated from cv. Golden Promise were not expressed in the studied barley cultivar Barke although showed primer specificity with their cloned DNA sequences",
author = "Kaczmarczyk, {Agnieszka Ewa} and Steve Bowra and Zoltan Elek and {\'E}va Vincze",
year = "2012",
doi = "10.1186/1471-2229-12-184",
language = "English",
volume = "12",
journal = "B M C Plant Biology",
issn = "1471-2229",
publisher = "BioMed Central Ltd.",
number = "184",

}

RIS

TY - JOUR

T1 - Quantitative RT-PCR based platform for rapid quantification of the transcripts of highly homologous multigene families and their members during grain development

AU - Kaczmarczyk, Agnieszka Ewa

AU - Bowra, Steve

AU - Elek, Zoltan

AU - Vincze, Éva

PY - 2012

Y1 - 2012

N2 - BackgroundCereal storage proteins represent one of the most important sources of protein for food and feed and they are coded by multigene families. The expression of the storage protein genes exhibits a temporal fluctuation but also a response to environmental stimuli. Analysis of temporal gene expression combined with genetic variation in large multigene families with high homology among the alleles is very challenging.ResultsWe designed a rapid qRT-PCR system with the aim of characterising the variation in the expression of hordein genes families. All the known D-, C-, B-, and gamma-hordein sequences coding full length open reading frames were collected from commonly available databases. Phylogenetic analysis was performed and the members of the different hordein families were classified into subfamilies. Primer sets were designed to discriminate the gene expression level of whole families, subfamilies or individual members. The specificity of the primer sets was validated before successfully applying them to a cDNA population derived from developing grains of field grown Hordeum vulgare cv. Barke. The results quantify the number of moles of transcript contributed to a particular gene family and its subgroups. More over the results indicate the genotypic specific gene expression.ConclusionsQuantitative RT-PCR with SYBR Green labelling can be a useful technique to follow gene expression levels of large gene families with highly homologues members. We showed variation in the temporal expression of genes coding for barley storage proteins. The results imply that our rapid qRT-PCR system was sensitive enough to identify the presence of alleles and their expression profiles. It can be used to check the temporal fluctuations in hordein expressions or to find differences in their response to environmental stimuli. The method could be extended for cultivar recognition as some of the sequences from the database originated from cv. Golden Promise were not expressed in the studied barley cultivar Barke although showed primer specificity with their cloned DNA sequences

AB - BackgroundCereal storage proteins represent one of the most important sources of protein for food and feed and they are coded by multigene families. The expression of the storage protein genes exhibits a temporal fluctuation but also a response to environmental stimuli. Analysis of temporal gene expression combined with genetic variation in large multigene families with high homology among the alleles is very challenging.ResultsWe designed a rapid qRT-PCR system with the aim of characterising the variation in the expression of hordein genes families. All the known D-, C-, B-, and gamma-hordein sequences coding full length open reading frames were collected from commonly available databases. Phylogenetic analysis was performed and the members of the different hordein families were classified into subfamilies. Primer sets were designed to discriminate the gene expression level of whole families, subfamilies or individual members. The specificity of the primer sets was validated before successfully applying them to a cDNA population derived from developing grains of field grown Hordeum vulgare cv. Barke. The results quantify the number of moles of transcript contributed to a particular gene family and its subgroups. More over the results indicate the genotypic specific gene expression.ConclusionsQuantitative RT-PCR with SYBR Green labelling can be a useful technique to follow gene expression levels of large gene families with highly homologues members. We showed variation in the temporal expression of genes coding for barley storage proteins. The results imply that our rapid qRT-PCR system was sensitive enough to identify the presence of alleles and their expression profiles. It can be used to check the temporal fluctuations in hordein expressions or to find differences in their response to environmental stimuli. The method could be extended for cultivar recognition as some of the sequences from the database originated from cv. Golden Promise were not expressed in the studied barley cultivar Barke although showed primer specificity with their cloned DNA sequences

U2 - 10.1186/1471-2229-12-184

DO - 10.1186/1471-2229-12-184

M3 - Journal article

VL - 12

JO - B M C Plant Biology

JF - B M C Plant Biology

SN - 1471-2229

IS - 184

ER -