Abstract
Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not ‘finished’. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full-length cDNA sequences.
Highlights
Messenger RNA sequence information is necessary for a variety of studies including mRNA expression, designing mRNA microarrays and genome annotation
We provide a general method for biologists experiencing difficulty in obtaining full-length cDNAs
As OC-17 protein is predominantly present in the adult hen uterus during eggshell formation, we collected a mature hen uterus sample to process for RNA-Seq
Summary
Messenger RNA sequence information is necessary for a variety of studies including mRNA expression, designing mRNA microarrays and genome annotation. Researchers generally apply mapping-based methods to detect mRNA structure for model organisms. This method will miss important genes that are located in gapped regions of the reference genome. Discovering the mechanisms that regulate biomineral formation will provide important insights for advances in human health, material sciences and biology [7]. 20 amino acids of OC-17 are found on the ChrUn in the chicken reference genome This is a relatively frequent case in biology where researchers know the amino acid sequence, have conducted many protein-based studies, but lack mRNA sequences for expression studies. We provide a general method for biologists experiencing difficulty in obtaining full-length cDNAs
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