The spatio-temporal expression analysis of parathyroid hormone like hormone gene provides a new insight for bone growth of the antler tip tissue in sika deer

Abstract

Objective: Parathyroid hormone like hormone (PTHLH), as an essential factor for bone growth, is involved in a variety of physiological processes. The aim of this study was to explore the role of PTHLH gene in the growth of antlers.

Methods: The coding sequence (CDS) of PTHLH gene cDNA was obtained by cloning in sika deer (Cervus nippon), and the bioinformatics was analyzed. The quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the differences expression of PTHLH mRNA in different tissues of the antler tip at different growth periods (early period, EP; middle period, MP; late period, LP).

Results: The CDS of PTHLH gene was 534 bp in length and encoded 177 amino acids. Predictive analysis results revealed that the PTHLH protein was a hydrophilic protein without transmembrane structure, with its secondary structure consisting mainly of random coil. The PTHLH protein of sika deer had the identity of 98.31%, 96.82%, 96.05%, and 94.92% with Cervus canadensis, Bos mutus, Oryx dammah and Budorcas taxicolor, which were highly conserved among the artiodactyls. The qRT-PCR results showed that PTHLH mRNA had a unique spatio-temporal expression pattern in antlers. In the dermis, precartilage, and cartilage tissues, the expression of PTHLH mRNA was extremely significantly higher in MP than in EP, LP (p<0.01). In the mesenchyme tissue, the expression of PTHLH mRNA in MP was significantly higher than that of EP (p<0.05), but extremely significantly lower than that of LP (p<0.01). The expression of PTHLH mRNA in antler tip tissues at all growth periods had approximately the same trend, that is, from distal to basal, it was first downregulated from the dermis to the mesenchyme and then continuously up-regulated to the cartilage tissue.

Conclusion: PTHLH gene may promote the rapid growth of antler mainly through its extensive regulatory effect on the antler tip tissue.

Keywords: Antlers; Characterization; Cloning; Gene Expression; Sika Deer.

Figure 1

Polymerase chain reaction amplification results of PTHLH gene. M, DL2000 Plus DNA Marker. a, amplification product. 1 to 5, positive detection results of cloning products. PTHLH, parathyroid hormone like hormone.

Figure 2

Structure of the encoded protein of the PTHLH gene in sika deer. (A) Nucleotide sequence and deduced amino acid sequence and secondary structure of PTHLH gene. * stop codon. (B) Tertiary structure of PTHLH protein. PTHLH, parathyroid hormone like hormone.

Figure 3

Bioinformatics prediction results of PTHLH protein. (A) Prediction results of hydrophilicity/hydrophobicity. (B) Prediction results of signal peptide. Sec/SPI represented secretory signal peptides transported by the Sec translocon and cleaved by Signal Peptidase I. Sec/SPI n, Sec/SPI h and Sec/SPI c represented the region structure of the Sec/SPI. CS, Cleavage site. (C) Prediction results of transmembrane structure. (D) Prediction results of phosphorylation sites. PTHLH, parathyroid hormone like hormone.

Figure 4

Differential expression of PTHLH mRNA in different tissues. (A) Dermis tissue. (B) Mesenchyme tissue. (C) Precartilage tissue. (D) Cartilage tissue. EP, early period. MP, middle period. LP, late period. PTHLH, parathyroid hormone like hormone. * 0.01<p<0.05. ** p<0.01.

Figure 5

Differential expression of PTHLH mRNA at different growth periods. (A) Early period. (B) Middle period. (C) Late period. PTHLH, parathyroid hormone like hormone. * 0.01<p<0.05. ** p<0.01.