藏系绵羊Smo基因的克隆测序及其功能的细胞水平验证

Cloning and sequencing of Smo gene of Tibetan sheep and primary identification of its functions at cellular level

实验目的是构建藏系绵羊Smoothened(Smo)基因的皮肤特异表达载体和干扰载体,并探索其在毛囊细胞中的调控机制.试验从成年藏系绵羊(n=5)皮肤中克隆了Smo基因,获得的编码区长度为2342 bp,编码779个氨基酸,与绵羊预测的Smo基因在序列上高度相似;试验构建了该基因含KAP6.1启动子的皮肤特异表达载体pCDsRed2-KS和3个干扰载体pcDNA6.2-GW/EmGFP-miRⅠ-Ⅲ;随后转染体外培养的人毛囊内根鞘细胞(HHIRSC),获得的转染效率均在约40%,转染48 h后从HHIRSC中提取总RNA,用定量PCR检测Smo基因及Hh通路中Gli1和Gsk3β的mRNA水平.结果显示,藏系绵羊Smo基因在转染表达载体的HHIRSC中获得超表达,与对照组相比,其mRNA水平提高约5900倍,同时Gli1、Gsk3β基因的mRNA水平也表现出极显著上调(P<0.01);构建的干扰载体转染HHIRSC后,对Smo基因的沉默效率均在98%以上,同时Gli1和Gsk3β的mRNA水平极显著下调(P<0.01).本研究提示藏系绵羊Smo基因可能通过Hh通路影响毛囊细胞功能.

The objective of this study was to construct skin-specific expression vector and interference vectors of Tibetan sheep Smoothened(Smo)gene,and explore its regulation mechanism in follicle cells.The Smo gene was cloned from skin samples of Tibetan sheep(n=5),the resulting coding sequence was 2342 bp in length,encoding 779 amino acids.This sequence showed high similarity with that of predicted sheep Smo gene;this experiment constructed skin-specific expression vector pCDsRed2-KS containing KAP6.1 promoter and three interfering vectors pcDNA6.2-GW/EmGFP-miRⅠ-Ⅲ;then these vectors were transfected to human hair follicle inner root sheath cells(HHIRSC)in vitro,and the transfection efficiencies were all about 40%.Total RNA was extracted from HHIRSC 48 h after transfection,and quantitative PCR was used to examine mRNA levels of Smo,Gli1 and Gsk3β in Hh pathway.The results showed that Smo gene was over expressed in HHIRSC transfected with pCDsRed2-KS vector,increased by approximately 5900 fold compared with control group,and both Gli1 and Gsk3β mRNA increased significantly(P<0.01);the silencing efficiencies of the three interfering vectors of Smo in transfected HHIRSC were all over 98%,and both Gli1 and Gsk3β mRNA levels reduced significantly(P<0.01).The present results suggest that the Smo gene of Tibetan sheep may influence functions of follicle cells through Hh signaling pathway.