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 γ-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.

Effects of Zn Fertilization on Hordein Transcripts at Early Developmental Stage of Barley Grain and Correlation with Increased Zn Concentration in the Mature Grain

“…In barley the proportion of the different hordein groups during grain development varies slightly but the amount of total hordein transcripts increased from 10 DAP onward and the B-hordein transcripts always appear as the highest proportion (more than 80%) compared to the other hordeins [33]. Zinc uptake rate of the wheat endosperm reaches its maximum level at 14 days after anthesis (DAA).…”

Zinc Blotting Assay for Detection of Zinc‐Binding Prolamin in Barley (Hordeum vulgare) Grain

“…It was not possible to separate B-and γ-hordein in 1-D gel because both had approximately similar molecular weight. However, because B-hordein is much more abundant (70-80% of total hordein) than γ-hordein (less than 5% of total hordein), the band in the 65 Zn autoradiography probably came from B-hordein, but that does not exclude the possibility that γ-hordein is capable of zinc ion binding as well (Shewry 1993;Kaczmarczyk et al 2012). Furthermore, both the radioactive labeling and DTZ staining assays showed correlation with zinc ion binding between barley lines differing in their B-hordein contents (Fig.…”

Basic and modern concepts on cholinergic receptor: A review

“…Diverse nAChR subtypes with various subunit combinations conferring differential selectivity for nicotinic drugs [32] , and also identified family of genes coding for proteins homologous to α subunit of the muscle nicotinic acetylcholine receptor in the rat genome. These genes are transcribed in the central and peripheral nervous systems in areas which are known to contain functional nicotinic receptors [33] . The role played by β2-containing neuronal nicotinic receptors (nAChRs) in mediating nicotine's side effects in the fetus and newborn.…”