Construction And Functional Analysis Of Skin-specific Expression And Interference Vectors Of Tibetan Sheep Smo Gene

The objective of this study was to construct Smo gene(one of the Gasdermin gene family members) skin-specific eukaryotic expression vector and interference vector of Tibetan sheep, and to explore possible regulation mechanism of Smo in follicle cells at cellular level and to provide a theoretical basis for further studying the feature of sheep Smo genes in hair follicle development and cycling. Adult Tibetan sheep was used to clone Smo gene from the skin tissue and to construct skin-specific eukaryotic expression vector and three interference vectors. Then transfected human hair inner root sheath cells in vitro using the constructed vectors. Real-time PCR was used to detect in the transfected cells the mRNA level of Smo gene of Tibetan sheep and Gli1 gene, Gsk3? gene, which are down stream genes of Hh signaling pathway. The main findings are as follows:(1) A 2342 bp coding sequence(CDS) of Smo gene of Tibetan sheep was obtained. Sequence alignments of CDS region showed that the Smo sequence of Tibetan sheep and predicted sequence of sheep Smo gene have three base differences, and one amino acid difference in protein sequence.(2) Based on the pCDsRed2-KI eukaryotic vector, a skin-specific eukaryotic expression vector pCDsR-KS containing Smo gene was constructed with KAP6.1 promoter. Three interference vectors were constructed with linear plasmid pcDNA 6.2-GW/EmGFP-miR.(3) The pCDsR-KS vector and three interference vectors were transfected into HHIRSC, respectively. RT-PCR detection of transfection results demonstrated that Tibetan sheep Smo gene was overexpressed in HHIRSC transfected with pCDsR-KS vector, also, Gli1 and Gsk3? gene expressions increased at mRNA levels(p<0.01). The three miRNA interference vectors showed over 98 % silence effect on Smo gene, leading to significantly decreased mRNA levels of Gli1 and Gsk3? genes(p<0.01).These results suggest that Tibetan sheep skin-specific eukaryotic expression vector pCDsR-KS and 3 interference vectors were successfully constructed, and it can be used to study the Smo gene functions at cellular level; In addition, Smo gene of Tibetan sheep might affect the follicle development or cycling through the Hh signaling pathway.