| The nucleotide sequence and tissue expression profiles of Tibetan sheep MSX2 gene, member of Homeobox gene family, and its effect on the expression of intracellular signaling molecules were analyzed in this study, aiming at providing information for exploring the influence of MSX2 gene in Tibetan sheep hair follicle development and other processes. Tissues of female Tibetan sheep(n=5) including heart, liver, spleen, lung, kindey, skin and fat were collected and total RNA was extracted from those samples. The coding region of MSX2 gene was cloned from the skin and its expression profile was compared among different tissues. Skin cell-specific expression vector of MSX2 gene was constructed and transferred into human hair inner root sheath cells(HHIRSC). Quantitative PCR and Western blot were used to examine the mRNA and protein expression levels of downstream target genes of MSX2 gene. The main experimental results are as follows:1. The MSX2 gene of Tibetan sheep was cloned and its coding region was 804 bp. Compared with the predicted sequence of sheep MSX2, the coding region of Tibetan sheep MSX2 gene showed 6 base differences and the similarity of two matching sequences was 99.25%. In addition, there were 6 base deletions in Tibetan sheep MSX2 gene. The deduced amino acid sequence of Tibetan sheep MSX2 gene contained 267 amino acids with theoretical molecular weight of 28.98 kD. The predicted theoretical isoelectric point of MSX2 gene of Tibetan sheep and sheep were both 10.32, however, there were three amino acid substitutions and two consecutive amino acid deletions in the deduced amino acid sequence of Tibetan sheep. The similarity of the deduced amino acid sequences between Tibetan sheep and sheep was 98.88%.2. Real-time quantitative PCR was used to analyze the expression levels of MSX2 gene in Tibetan sheep heart, liver, spleen, lung, kidney, skin and fat. The results showed that the expression of MSX2 gene in skin was significantly greater than other tissues(P<0.01), suggesting that MSX2 gene may be associated with Tibetan sheep hair growth or traits.3. Skin cell-specific expression vector pCDsR-KM was constructed by ligating MSX2 gene to downstream of KAP6.1 promoter, and then inserting the restructuring fragment into the basic vector pCDsRed2. The recombinant eukaryotic expression vector was transiently transfected into human hair follicle root sheath cells(HHIRSC), with transfection efficiency of about 10%. The HHIRSC were sorted by flow cytometry and the successful transfection cells were obtained.4. Real-time PCR results showed that MSX2 gene was overexpressed for about 9000 times and the Lef1, Foxn1, Notch1 and Notch2 genes, downstream target genes of MSX2, were significantly upregulated in the transfected HHIRSC sorted by flow cytometry. Furthermore, the protein expression levels of Notch1 and Notch2 were detected by Western blot and the results revealed that the two proteins were also significantly upregulated in expermential group, suggesting that MSX2 gene might regulate the skin follicle development and cyclical change through Wnt, Notch, MSX2/Foxn1/hair keratin and other signaling pathways. |
