Screening Differentially Expressed Genes In Different Pattern Individuals Of Hu-sheep And Major Gene Functional Verification

In this study, we focused on the hu-sheep individuals and screened differentially expressed genes of new born Hu-sheep’s skin tissue by Agilent gene chip, discussed the histological characteristics of hu-sheep’s dorsal skin follicle by histology and microscpic observation method; we collected15hu-sheep individuals(large waves、 samll waves、medium waves), Histological features of the new born hu-sheep’s skin follicles, combined with fluorescence quantitative PCR technology was used to detect the correlation between the expression of7genes in different individuals and hair follicle development, in order to choose futher functional verification gene-BMP7; Gene cloning method was used to clone full-length cDNA sequence of sheep BMP7and biological analysis software was used to analyze the BMP7sequence homology between sheep and other species, physicochemical properties, secondary and tertiary structure. Constructed pEGFP-Cl-BMP7eukaryotic expression vector to reveal the regulatory mechanism of BMP7gene in hair follicle development and laid a theoretical foundation on futher understand of the mechanism in different pattern formation,The results were followed as these:1、Approximately137genes were found to be differentially expressed among large-and small-wave follicles using gene chip technology. Gene ontology analysis indicated that some of these genes are involved in cell proliferation, differentiation, apoptosis, development, metabolization, and the immune response. The abnormal expression of BMP7, MMP2, SNAI1, SFXN1, CDKNIC, MT3, and POU1F1showed that they may participate in the formation of hair follicles and so may produce the dorsal pattern. Histological studies showed that the P/S of hu-sheep hair follicle tissue, small waves was higher than that in large groups and had a significantly difference (P<0.05). The total number of hair follicles was no significantly difference between two groups, but small waves group more than large waves group. The diameter of small waves’primary hair follicle smaller than large waves group and had a significantly difference (P<0.05). Small waves group was smaller than large waves group in the dimater of secondary hair follicle and had a extremely significant difference (P<0.01)2、Among the genes studied MMP2, BMP7and SFXN1genes were expressed significantly different in large, medium and small waves (P<0.05). The expression of MMP2had a significant positive correlation with secondary follicle in large waves (P<0.05), while the expression of BMP7had a significant correlation with primary follicle diameter in small waves, and extreme significant positive correlation with the number of small waves’secondary follicles (P<0.01). The expression of SFXN1had a significant positive correlation with small waves’primary follicle diameter and extreme significant positive correlation with secondary follicle diameter. Although other genes associated with hair follicles, but their expression in large, medium, small waves were not significant. It inferred that BMP7, MMP2, SFXN1genes could be as the important candidate genes for early breeding.3、The complete full-length CDS of BMP7gene was1296bp, which encoded431amino acids, the molecular weight and theoretical isoelectric point p1was49316.9Da and7.75, included unstable proteins. Nucleotide sequence analysis showed the amino acid sequence of BMP7gene was high homology with bovine、human and dogs, and with zebrafish granosa relatively low. There was a TGFβ superfamily domains in amino acids330-431, indicated that it has the attribute of TGFβ family. The subcellular localization results indicated that BMP7gene located in the cell, synthesis in the intracellular and then secretion into the extracellular for function, it belonging secreted proteins. Function prediction analysis showed that BMP7proteins played an important role in the translation process、fatty acid metabolism, and also played a key role in cellular processes、central intermediary metabolism.4、It was confirmed that the sheep BMP7CDS was correctly inserted into eukaryotic expression vector by PCR, restriction digestion and sequencing. This study settled a experimental basis for exproing BMP7regulation mechanism in the process of hair follicle cells proliferation and differentiation.