Stable expression of Y-box protein 1 gene in early development of the abalone Haliotis diversicolor

Abstract

Abalone animals are import models for the study of the early development of marine invertebrates. However, systematical evaluations of internal control genes (ICG) have seldomly been performed. In this study, ten candidate genes were cloned and surveyed for their stability throughout the early developmental period of H. diversicolor using qPCR. In a period from fertilized egg to postlarva, three genes, Y-box protein 1 (YB1), ornithine decarboxylase antizyme 1 (OAZ1) and eukaryotic translation initiation factor 5A (EIF5A), were found to be the most stable and could be used as ICGs. It is suggested that using two genes jointly, such as YB1 and OAZ1, could be sufficiently reliable to normalize the temporal dynamics of other genes. Normalized by YB1/OAZ1, some rough features of early development of a small abalone were characterized. This is the first report of the temporal dynamics of metabolic activities and overall mRNA abundance of abalone animals in early stages. It is also the first time the multi-functional gene YB1 has been described as an internal control for early developmental biology studies. Phylogeny and function of YB1 are further discussed.

Pyrosequencing of Haliotis diversicolor Transcriptomes: Insights into Early Developmental Molluscan Gene Expression

“…The first part of the verification was involved evaluating the stably expressed genes during the early developmental stages by using 9 genes. Y-box protein 1 ( YB1 ), ornithine decarboxylase antizyme 1 ( OAZ1 ) and eukaryotic translation initiation factor 5A ( EIF5A ) qualified as internal control genes (ICG) after such an evaluation [35]. The second part of the verification was to calibrate the qPCR relative expression levels of another 20 scaffolds/contigs by applying the reliable ICGs and to then evaluate the correlations between the two types of temporal dynamics using Pearson’s correlation test (Figure 5).…”

“…Template cDNA preparation and primer quality controls were performed as internal control gene (ICG) evaluations [35]. In brief, another batch of embryonic/larval samples was prepared and collected from the same abalone farm.…”

“…The PCR conditions were as follows: 95°C for 3 minutes, (95°C for 10 seconds, 55°C for 15 seconds, 72°C for 15 seconds) × 45 cycles. The relative expression levels of each gene for the different developmental stages were normalized to the Y-box protein 1 gene ( YB1 ) and ornithine decarboxylase antizyme 1 gene ( OAZ1 ), which have been verified as reliable ICGs in previous studies [35]. To evaluate whether the relative expression levels of the qPCR and 454 corresponded, the 454 data was also normalized using the 454 expression data for YB1 / OAZ1 and then compared with the qPCR data using Pearson’s correlation tests in Microsoft Excel.…”

A-to-I editing of Malacoherpesviridae RNAs supports the antiviral role of ADAR1 in mollusks

“…qRT-PCR reactions were carried out starting from 1 μl of cDNA in 15 μl of final reaction mixture using the DyNAmo HS SYBR Green qPCR kit (Thermo Scientific, Waltham, USA). The housekeeping gene Elongation factor 1 alpha (EF1-α) was chosen as reference for C. gigas , whereas the Y-box binding protein 1 was used for H. diversicolor , since it was verified as reliable housekeeping gene in a previous study [100]. OsHV-1 ORF104 and its AbHV-1 homolog, ORF68, were used as proxy to determine the overall viral transcription according to their robust expression trend previously reported [76, 78].…”

Involvement of Antizyme Characterized from the Small Abalone Haliotis diversicolor in Gonadal Development

“…Research on ODC AZ from abalone is limited. The AZ gene was validated to be used as a single nucleotide polymorphism marker to evaluate the loss of genetic diversity due to hatchery selection [ 53 ] or internal control gene [ 54 ] in abalone.…”