摘要
[目的]克隆获得和田羊皮肤角蛋白79(keratin 79,KRT79)基因,探究雄激素对和田羊皮肤结构的影响,了解KRT79在雄激素影响和田羊皮肤结构过程中发挥的作用,为深入研究KRT79在绵羊皮肤中的功能及雄激素对KRT79基因表达调控的机制奠定基础。[方法]选取15只2岁和田母羊,随机分为5组,采用肌内注射的方法对对照组和田羊注射玉米油,对4个试验组和田羊分别注射1、2、3、4 mg/kg BW睾酮,每3天注射一次,共处理42 d,在第42天采集和田羊背部皮肤组织。从对照组和田羊皮肤中提取总RNA,PCR扩增、克隆KRT79基因CDS区序列并测序。利用DNAStar软件对和田羊KRT79基因序列与绵羊等物种相应的基因序列进行比对,采用Mega 5.0软件绘制各物种KRT79基因系统进化树。使用生物信息学在线网站分析KRT79蛋白的理化性质及结构特征。通过HE染色观察睾酮处理的和田羊皮肤结构变化,使用实时荧光定量PCR和Western blotting检测分析不同浓度睾酮处理的和田羊皮肤中KRT79基因和蛋白表达量的差异,并通过免疫荧光染色探究睾酮对KRT79蛋白在和田羊皮肤中表达分布的影响。[结果]和田羊KRT79基因CDS区序列长1 614 bp,与绵羊的相似性为99.9%。系统进化树分析表明,和田羊KRT79基因与绵羊的进化关系最近,与小鼠的进化关系最远。和田羊KRT79基因编码537个氨基酸,是一种不稳定的亲水性蛋白,KRT79蛋白的二级结构主要由α-螺旋和无规则卷曲组成。形态学分析结果表明,睾酮处理后和田羊皮肤中皮脂腺面积增大,数量变多。实时荧光定量PCR、Western blotting和免疫荧光结果表明,睾酮处理后和田羊皮肤中KRT79基因和蛋白的表达水平显著高于对照组(P<0.05),KRT79蛋白主要分布于和田羊皮肤的皮脂腺、表皮和毛囊区域。[结论]和田羊KRT79基因CDS区长1 614 bp,是一种不稳定的亲水性蛋白。雄激素刺激能显著促进和田羊皮肤的皮脂腺面积增大和数量变多,并促进皮肤中KRT79基因和蛋白的表达。研究结果为深入探讨雄激素对和田羊皮肤的影响及其作用机制提供了理论依据,同时为解决由雄激素引起的皮肤脂质代谢异常和角蛋白失调疾病治疗方法的研究提供了参考。
[Objective]This study was aimed to clone and obtain the keratin 79(KRT79)gene from the skin in Hetian sheep,investigate the effects of androgens on the skin structure of Hetian sheep,and understand the role of KRT79 in the process of influencing the skin structure of Hetian sheep through androgens,so as to lay the foundation for in-depth research on the function of KRT79 in sheep skin and the mechanism of androgen on the expression and regulation of KRT 79 gene.[Method]Fifteen 2-year-old Hetian ewes were randomly divided into five groups with three in each group.Corn oil was injected into Hetian sheep in control group by intramuscular injection,while testosterone at doses of 1,2,3,and 4 mg/kg BW were injected into Hetian sheep in four experimental groups every three days for a total of 42 days.On the 42nd day,the back skin tissue was collected.Total RNA was extracted from the skin of Hetian sheep in control group,and the KRT 79 gene CDS sequence was amplified by PCR,cloned and sequenced.The obtained KRT 79 gene sequence of Hetian sheep was compared with the corresponding gene sequences of Ovis aries and other species using DNAStar software,and the phylogenetic tree of KRT 79 gene of each species was constructed by Mega 5.0 software.The physicochemical property and structural characteristic of KRT79 protein were analyzed by bioinformatics online website.HE staining was used to observe the changes in the skin structure of Hetian sheep treated with testosterone.Real-time quantitative PCR and Western blotting were used to detect and analyze the difference of KRT 79 gene and protein expression in the skin of Hetian sheep treated with different concentrations of testosterone.Immunofluorescence staining was used to explore the effect of testosterone on the expression distribution of KRT79 protein in the skin of Hetian sheep.[Result]The CDS region sequence length of KRT 79 gene in Hetian sheep was 1614 bp.The similarity of KRT 79 gene sequence was 99.9%between Hetian sheep and Ovis aries.Phylogenetic tree analysis demonstrated that KRT 79 gene had the closest evolutionary kindred with Ovis aries and the farthest evolutionary kindred with Mus musculus.KRT79 gene encoded 537 amino acids,which was an unstable hydrophilic protein,and the secondary structure of KRT79 protein was mainly comprised of alpha helix and random coil.The results of morphological analysis showed that the area of sebaceous glands in the skin of Hetian sheep expanded after testosterone treatment,and the number raised.Real-time quantitative PCR,Western blotting and immunofluorescence results indicated that the expression of KRT 79 gene and protein in the skin of Hetian sheep were significantly higher than that in control group after testosterone treatment(P<0.05).KRT79 protein was mainly distributed in the sebaceous glands,epidermis,and hair follicle areas of the skin in Hetian sheep.[Conclusion]The CDS region of KRT 79 gene in Hetian sheep was 1614 bp,and was an unstable hydrophilic protein.Androgen stimulation could significantly promote an increase in the area and quantity of sebaceous glands in the skin of Hetian sheep,and promote the expression of KRT 79 gene and protein in the skin.This results provided a theoretical basis for further exploring the effects and mechanisms of androgens on the skin in Hetian sheep,and also provided a reference for the development of treatment methods for skin lipid metabolism abnormalities and keratin imbalance diseases caused by androgens.
作者
冯稚雅
彭婉婉
张建萍
武振辉
张楠
李树伟
史瑞军
FENG Zhiya;PENG Wanwan;ZHANG Jianping;WU Zhenhui;ZHANG Nan;LI Shuwei;SHI Ruijun(Xinjiang Production&Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin,College of Life Science and Technology,Tarim University,Alar 843300,China)
出处
《中国畜牧兽医》
北大核心
2025年第2期562-573,共12页
China Animal Husbandry & Veterinary Medicine
基金
塔里木大学校长基金(TDZKBS202309)
“天池英才”引进计划青年博士项目
天山英才-科技创新领军人才培育项目(2023TSYCLJ0057)。
作者简介
冯稚雅,E-mail:1992909175@qq.com;通信作者:李树伟,E-mail:yfzx_406@126.com;通信作者:史瑞军,E-mail:shiruijun2021@163.com。
