奶山羊p27基因克隆分析及其对乳腺上皮细胞增殖的影响  

Cloning and analysis of dairy goat p27gene and its effects on the proliferation of mammary epithelial cells

作　　者：岳子婷 黄江涛[1] 宁勇 宗学阳 张华文 史怀平[1] YUE Ziting;HUANG Jiangtao;NING Yong;ZONG Xueyang;ZHANG Huawen;SHI Huaiping(College of Animal Science and Technology,Northwest A&F University,Yangling 712100,Shaanxi,China)

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《陕西师范大学学报（自然科学版）》2022年第5期81-89,共9页Journal of Shaanxi Normal University：Natural Science Edition

基　　金：陕西省创新能力支撑计划(2021TD-31);西安市科技计划(20NYYF0030)。

摘　　要：细胞周期依赖性激酶抑制蛋白B(cyclin dependent kinase inhibitors B,CDKNIB,即p27)可以抑制细胞周期蛋白-细胞周期蛋白依赖性激酶复合物,阻滞细胞周期进程。通过克隆获得奶山羊p27基因序列并阐明其编码蛋白的生物学特性,利用RNAi干扰技术和基因过表达技术沉默和上调奶山羊乳腺上皮细胞中p27基因的表达,以探究p27基因对乳腺上皮细胞增殖的影响。以西农萨能奶山羊乳腺组织为实验材料,扩增并克隆出奶山羊p27基因CDS全长序列,其全长597 bp,编码198个氨基酸。对p27基因编码蛋白进行生物信息学分析,发现其为亲水性不稳定蛋白,无跨膜结构。利用实时荧光定量PCR检测奶山羊p27基因mRNA在不同时期乳腺组织中的表达情况,发现p27基因在泌乳期的表达量高于干奶期。将构建的p27基因真核表达质粒转染奶山羊乳腺上皮细胞,使用CCK-8、Edu染色及蛋白质印迹法(Western blot)检测p27基因对乳腺上皮细胞增殖的影响,发现过表达p27基因抑制了乳腺上皮细胞的增殖和DNA合成,并使得CDK2蛋白表达水平升高(P<0.05)。上述结果提示p27可作为负调节因子参与乳腺上皮细胞增殖,研究结果为p27调控乳腺发育的分子机理提供了基础资料。Cyclin dependent kinase inhibitors B(CDKNIB,namely p27)inhibits cyclin-cyclin dependent kinase complexes and halts cell cycle progression.After cloning p27gene sequence of dairy goat and elucidating its biological characteristics,RNA interference and gene overexpression techniques were used to explore the function of p27gene on the proliferation of mammary epithelial cells.Taken mammary tissue from Xinong Saanen dairy goats as experimental material,the whole CDS sequence of p27gene was cloned and the bioinformatics analysis of p27gene coding sequence was conducted.Results showed that the complete CDS region of p27gene was 597 bp,encoding 197 amino acids,and p27 protein was a hydrophilic unstable protein without transmembrane spiral structure.The expression of p27gene in mammary tissue of different stages was measured by RT-PCR,and higher expression level was detected at lactation stage compared with that at dry period.The eukaryotic expression plasmid of p27gene was transfected into mammary epithelial cells,and its effects on the proliferation of mammary epithelial cells were evaluated by CCK-8,Edu staining and Western blot.Results showed that overexpression of p27gene inhibited the proliferation and DNA synthesis of mammary epithelial cells,and increased the expression of CDK2(P<0.05).The above results suggest that p27 is a negative regulator in the proliferation of mammary epithelial cells,which provides basic data for further investigating the molecular mechanism of p27 on mammary gland development.

关 键 词：奶山羊 P27基因 克隆 细胞增殖 

分 类 号：Q812[生物学—生物工程]