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Molecular analysis of CIB4 gene and protein in Kermani sheep.

ABSTRACT: The human calcium- and integrin-binding protein (CIB) family is composed of CIB1, CIB2, CIB3, and CIB4 proteins and the CIB4 gene affects fertility. Kermani sheep is one of the most important breeds of Iranian sheep breeds. The aim of this study was to analyze for the first time molecular characteristics of the CIB4 gene and protein in Kermani sheep. Different tissues were collected from the Kermani sheep and real time PCR was performed. The PCR products were sequenced, comparative analyses of the nucleotide sequences were performed, a phylogenetic tree was constructed, and different characteristics of CIB4 proteins were predicted. Real time PCR results showed that the CIB4 gene is expressed only in testis of Kermani sheep. The cDNA nucleotide sequence was identical with small tail Han sheep, cattle, goat, camel, horse, dog, mouse and human, respectively 100, 99, 99, 98, 98, 96, 96, and 96%. Hence, it can be suggested that the CIB4 gene plays a role in male fertility. Based on the phylogenetic analysis, sheep CIB4 gene has a close relationship with goat and cattle first, and then with camel and whale. Although we demonstrated that CIB4 is a testis-specific gene, expressed only in the testis and it interacts with other proteins, the mechanisms by which CIB4 expression is regulated need to be elucidated.

SUBMITTER: Mohammadabadi MR

PROVIDER: S-EPMC5597282 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Molecular analysis of CIB4 gene and protein in Kermani sheep.

Mohammadabadi M R MR Jafari A H D AHD Bordbar F F

Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas 20170912 11

The human calcium- and integrin-binding protein (CIB) family is composed of CIB1, CIB2, CIB3, and CIB4 proteins and the CIB4 gene affects fertility. Kermani sheep is one of the most important breeds of Iranian sheep breeds. The aim of this study was to analyze for the first time molecular characteristics of the CIB4 gene and protein in Kermani sheep. Different tissues were collected from the Kermani sheep and real time PCR was performed. The PCR products were sequenced, comparative analyses of t ...[more]

PMID: 28902924

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Project description:BackgroundBlack 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.MethodsIn 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.ResultsThe 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.ConclusionsThe 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.

Project description:Background:Ye Mule Aries sheep is one of the most important sheep breeds in Xinjiang, China. This breed is well adapted to harsh environmental conditions and displays strong disease resistance, fast growth, and high cold tolerance. To analyze the clonal expression and immunogenicity of the Ye Mule Aries sheep inhibin gene, total RNA was extracted from sheep ovarian tissue and used as a template to generate a eukaryotic expression vector and study inhibin immunogenicity. Methods:Primers were designed to amplify the inhibin A gene via polymerase chain reaction and the amplified product was cloned between the ScalI and EcoRI restriction sites of the expression vector pEGFP-N1 to construct a recombinant plasmid, pEGFP-INH?. Following the validation of successful cloning, the pEGFP-INH? plasmid was transfected into BHK cells to verify expression in eukaryotes and subsequently utilized as an antigen in rabbits. Rabbits were tested for anti-inhibin antibodies and serum follicle-stimulating hormone (FSH) concentrations. Results:The analysis of the INH? gene sequence revealed that INH? is 1109 bp long and is translated to an approximately 40 KDa protein. Bioinformatics approach indicated that the INH? gene is highly conserved between organisms. Immunization with the eukaryotic expression vector, pEGFP-INH?, which expresses the INH? gene elicited immune response and generatigeneration on of anti-INH? antibody. The antibody had a significant regulatory effect on the serum concentration of FSH in rabbits and led to higher levels of FSH, indicating increased ovary function. Conclusions:The present work resulted in a successful construction of eukaryotic expression plasmid pEGFP-INH? and verified the immunogenicity of this highly conserved protein. Further, the expression of pEGFP-INH? was shown to have a significant impact on the secretion of FSH, indicating a potential regulatory role in ovarian function. In conclusion, our current findings can serve as a working model for studying the effect of INH? on the breeding performance of Ye Mule Aries sheep, providing a novel strategy to improve their reproduction rates.

Dataset Information

Molecular analysis of CIB4 gene and protein in Kermani sheep.

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Molecular analysis of CIB4 gene and protein in Kermani sheep.

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