title: Integrating de novo transcriptome assembly and cloning to obtain chicken Ovocleidin-17 full-length cDNA.
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.
authors.affiliation: National Engineering Laboratory for Animal Breeding and MOA Key Laboratory of Animal Genetics and Breeding, Department of Animal Genetics and Breeding, China Agricultural University, Beijing, China.
authors.forename: Quan
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: National Engineering Laboratory for Animal Breeding and MOA Key Laboratory of Animal Genetics and Breeding, Department of Animal Genetics and Breeding, China Agricultural University, Beijing, China.
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.affiliation: Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Ya'an, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhu
authors.affiliation: National Engineering Laboratory for Animal Breeding and MOA Key Laboratory of Animal Genetics and Breeding, Department of Animal Genetics and Breeding, China Agricultural University, Beijing, China.
authors.forename: ZhongHua
authors.initials: Z
authors.lastname: Ning
authors.affiliation: Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada.
authors.forename: Maxwell T
authors.initials: MT
authors.lastname: Hincke
authors.affiliation: National Engineering Laboratory for Animal Breeding and MOA Key Laboratory of Animal Genetics and Breeding, Department of Animal Genetics and Breeding, China Agricultural University, Beijing, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Yang
authors.affiliation: National Engineering Laboratory for Animal Breeding and MOA Key Laboratory of Animal Genetics and Breeding, Department of Animal Genetics and Breeding, China Agricultural University, Beijing, China.
authors.forename: ZhuoCheng
authors.initials: Z
authors.lastname: Hou
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002119:Calcium Carbonate / Q000378:metabolism; D002645:Chickens / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D004527:Egg Proteins / Q000235:genetics* / Q000378:metabolism; D004528:Egg Shell / Q000737:chemistry / Q000254:growth & development / Q000378:metabolism*; D005260:Female; D018507:Gene Expression Regulation, Developmental; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D059467:Transcriptome; D014599:Uterus / Q000254:growth & development / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D004527:Egg Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C095273:ovocleidin 17; D002119:Calcium Carbonate