Zhang Quan, Liu Long, Zhu Feng, Ning ZhongHua, Hincke Maxwell T, Yang Ning, Hou ZhuoCheng
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.
Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Ya'an, China.
PLoS One. 2014 Mar 27;9(3):e93452. doi: 10.1371/journal.pone.0093452. eCollection 2014.
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.
高效获取目标基因的全长cDNA是功能研究和探索基因变异的关键步骤。然而，几乎所有已测序的家畜基因组都尚未“完成”。许多功能重要的基因位于这些缺口区域。对于仅存在部分氨基酸/EST序列的情况，获取全长cDNA可能会很困难。在本研究中，我们报告了一种获取全长cDNA的通用流程，并以与鸡蛋壳生物矿化相关的一个重要基因（卵壳蛋白-17，OC-17）为例说明了该方法。鸡OC-17是控制和调节碳酸钙在钙化蛋壳层沉积的最佳候选基因之一。OC-17蛋白已被纯化、测序，并且其三维结构已得到解析。然而，由于mRNA序列未知且当前鸡基因组中不存在该基因，研究人员仍然无法进行与OC-17 mRNA相关的研究。我们使用RNA-Seq获取成年母鸡子宫的整个转录组，然后通过生物信息学分析进行从头转录组组装以获得候选OC-17转录本。基于该序列，我们使用RACE和PCR克隆方法成功获得了全长OC-17 cDNA。还进行了OC-17 mRNA的时空表达分析，以证明OC-17在成年母鸡产蛋周期中主要在子宫中表达，而在未成熟发育阶段几乎不表达。在产弱蛋壳和强蛋壳的母鸡中观察到OC-17在子宫中的表达差异，证实了其在蛋壳矿化调节中的重要作用，并为蛋壳质量参数的遗传选择提供了新工具。本研究是首个报道全长OC-17 cDNA序列的研究，为与OC-17 mRNA相关的研究奠定了基础。我们为在获取候选基因全长cDNA序列方面遇到困难的生物学家提供了一种通用方法。
