HGTP基因与羊毛细度的相关性分析及其启动子活性研究

本研究旨在构建高甘氨酸－酪氨酸蛋白（HGTP）启动子荧光素酶报告基因载体并验证其活性，阐明HGTP基因在萨福克羊和中国美利奴羊皮肤毛囊组织中的表达差异与羊毛经济性状的关系。运用PCR方法扩增HGTP基因启动子序列片段，克隆至pGL3-Basic载体中构建重组质粒，并通过双酶切验证和测定核酸序列鉴定；转染皮肤成纤维细胞并检测报告基因的活性；测定羊毛的纤维直径、长度的数据，利用实时荧光定量PCR分析HGTP基因在不同品种绵羊的皮肤毛囊组织中的表达情况。结果显示，萨福克羊与中国美利奴羊的羊毛纤维直径及自然长度差异极显著（P<0.01），HGTP基因表达水平与羊毛纤维直径存在极显著的高度正相关（P<0.01），与羊毛自然长度呈现显著的中等负相关（P<0.05），而羊毛直径与自然长度之间存在极显著的高度负相关（P<0.01）。HGTP基因在萨福克羊和中国美利奴羊皮肤组织中的表达水平差异极显著（P<0.01）。启动子活性荧光素酶检测结果显示，KAP6.1、KAP7和KAP8.1启动子元件在绵羊皮肤成纤维细胞和3T3细胞中均有表达，且在绵羊成纤维细胞中的活性极显著高于在3T3细胞中的活性（P<0.01）。HGTP基因可以作为研究羊毛纤维直径、自然长度的候选基因。构建不同长度的表达载体，转染皮肤成纤维细胞，获得的2个启动子元件都能驱动外源基因在体外细胞水平表达。绵羊HGTP基因启动子的功能研究为调控羊毛发育提供了理论依据。

Study on the Correlation Between HGTP Gene and Wool Fineness and the Activity of HGTP Gene Promoter

This study was aimed to construct a high glycine-tyrosine protein (HGTP) promoter luciferase reporter vector,verify its activity,and elucidate the relationship between the expression difference of HGTP gene in the skin and hair follicle tissues of Suffolk and Chinese Merino sheep and the economic traits of wool.The HGTP gene promoter fragment was amplified by PCR and cloned into pGL3-Basic vector to construct a recombinant plasmid,which was verified by double enzyme digestion and nucleic acid sequence identification.Skin fibroblasts were transfected and the activity of the reporter gene was detected.The fiber diameter and length of the wool were measured,and the expression of HGTP gene in the hair follicle tissues of different breeds of sheep were analyzed by Real-time PCR.The results showed that the differences between wool fiber diameter and natural length of wool in Suffolk and Chinese Merino sheep were extremely significant (P<0.01).There was a highly significant positive correlation between the expression of HGTP gene and wool fiber diameter (P<0.01),a significant medium negative with the natural length of wool (P<0.05),and there was a very significant high negative correlation between wool diameter and natural length (P<0.01).The expression of HGTP gene in skin tissues of Suffolk and Chinese Merino sheep were extremely significant (P<0.01).The promoter activity luciferase assay showed that the KAP6.1,KAP7 and KAP8.1 promoter elements were expressed in both sheep skin fibroblasts and 3T3 cells,and the activity of the three promoter elements in sheep fibroblasts were extremely significantly higher than that in 3T3 cells (P<0.01).HGTP gene could be used as a candidate gene for studying the diameter and natural length of wool fibers.Expression vectors of different lengths were constructed and transfected into skin fibroblasts,both promoter elements were obtained to drive the expression of foreign genes at the cellular level in vitro.The functional study of HGTP gene promoter in sheep provided a theoretical basis for regulating wool development.