Molecular cloning and characterization of the endothelin 3 gene in black bone sheep

Hesham Y A Darwish; Yuanyuan Zhang; Kai Cui; Zu Yang; Deping Han; Xianggui Dong; Huaming Mao; Weidong Deng; Xuemei Deng

doi:10.1186/s40104-018-0272-y

Molecular cloning and characterization of the endothelin 3 gene in black bone sheep

J Anim Sci Biotechnol. 2018 Jun 25:9:57. doi: 10.1186/s40104-018-0272-y. eCollection 2018.

Authors

Hesham Y A Darwish^{1

2}, Yuanyuan Zhang¹, Kai Cui¹, Zu Yang¹, Deping Han^{1

3}, Xianggui Dong¹, Huaming Mao⁴, Weidong Deng⁴, Xuemei Deng¹

Affiliations

¹ 1National Engineering Laboratory for Animal Breeding and Key Laboratory of Animal Genetics, Breeding, and Reproduction of the Ministry of Agriculture, China Agricultural University, Beijing, 100193 China.
² Animal Production Research Institute, Agricultural Research Center, Ministry of Agriculture and Land Reclamation, Giza, 12618 Egypt.
³ 3College of Veterinary Medicine, China Agricultural University, Beijing, 100193 China.
⁴ 4College of Animal Science and Technology, Yunnan Agricultural University, Kunming, 650201 China.

Abstract

Background: Black bone sheep was first discovered in Yunnan province of China in 1970, with unique black pigmentation on the body and internal organs. Endothelin 3 (EDN3) has been known as a key gene causing hyperpigmentation in black bone chicken, the Silky fowl.

Methods: In this study, EDN3 was employed as a candidate gene for regulating black color pigmentation. First, EDN3 was cloned from sheep to obtain the full-length cDNA by using the rapid amplification of cDNA ends (RACE). Genomic EDN3 was screened and a total of thirty predicted single nucleotide polymorphisms (SNPs) were genotyped for allele and genotype frequency analysis in a case-control study involving two black bone sheep populations. Genomic copy number analysis of EDN3 in sheep was conducted to measure the variation in copy number. EDN3 expression levels were observed among the groups in adult liver, lymph node, and kidney tissues, as well as embryo kidney samples. Also, among the tissues of black bone and non-black bone sheep.

Results: The size of the full-length cDNA was 1,578 bp, which included 426 bp of 5'-untranslated region (5'-UTR), an open reading frame (ORF) of 639 bp encoding a protein of 212 amino acids, and a 3'-UTR of 513 bp. Genotype and allele frequencies of all the discovered SNPs were found insignificantly different in black bone and non-black bone sheep (P > 0.05). Genomic copy number analysis of EDN3 in sheep revealed no significant difference between the two sheep groups. No significant variations were found in the adult liver and kidney embryo samples. However, the expression in lymph node and kidney tissue was significantly higher in black bone sheep than that in non-black bone sheep (P < 0.05). Significant variations in the EDN3 expression levels were observed among the tissues of non-black bone sheep.

Conclusions: The findings of the present study indicate that unlike in Silky chickens, EDN3 is not responsible for hyperpigmentation but may play a key functional role in immune and excretory systems of black bone sheep.

Keywords: Black pigmentation; Expression level; SNP; Sequence analysis.