Cloning and Expression of FSHb Gene and the Effect of FSHβ on the mRNA Levels of FSHR in the Local Chicken

Zhao, L. H.; Chen, J. L.; Xu, H.; Liu, J. W.; Xu, R. F.

doi:2010.23.3.292

Article

Animal Breeding and Genetics

Asian-Australasian Journal of Animal Sciences 2010;23(3): 292-301.
https://doi.org/10.5713/ajas.2010.90473 Published online February 22, 2010.

Cloning and Expression of FSHb Gene and the Effect of FSHβ on the mRNA Levels of FSHR in the Local Chicken

L. H. Zhao, J. L. Chen, H. Xu, J. W. Liu, R. F. Xu

Abstract

Follicle-stimulating hormone (FSH) is a pituitary glycoprotein hormone that is encoded by separate alpha- and beta-subunit genes. It plays a key role in stimulating and regulating ovarian follicular development and egg production in chicken. FSH signal transduction is mediated by the FSH receptor (FSHR) that exclusively interacts with the beta-subunit of FSH, but characterization of prokaryotic expression of the FSHb gene and its effect on the expression of the FSHR gene in local chickens have received very little attention. In the current study, the cDNA fragment of the FSHb gene from Dagu chicken was amplified using reverse transcription polymerase chain reaction (RT-PCR), and inserted into the pET-28a (+) vector to construct the pET-28a-FSHb plasmid. After expression of the plasmid in E. coli BL21 (DE3) under inducing conditions, the recombination protein, FSH subunit, was purified and injected into the experimental hens and the effect on the mRNA expression levels of the FSHR gene was investigated. Sequence comparison showed that the coding region of the FSHb gene in the local chicken shared 99%-100% homology to published nucleotides in chickens; only one synonymous nucleotide substitution was detected in the region. The encoded amino acids were completely identical with the reported sequence, which confirmed that the sequences of the chicken FSHb gene and the peptides of the FSH subunit are highly conserved. This may be due to the critical role of the normal function of the FSHb gene in hormonal speciﬁcity and regulation of reproduction. The results of gene expression revealed that a recombinant protein with a molecular weight of about 19 kDa was efficiently expressed and it was identified by Western blotting analysis. After administration of the purified FSHβ protein, significantly higher expression levels were demonstrated in uterus, ovary and oviduct samples (p<0.05). These observations suggested that the expressed FSH protein possesses biological activity, and has a potential role in regulation of reproductive physiology in chickens.

Keywords: Chicken; FSHb Gene; Expression; FSH-receptor