牦牛AQP1基因克隆及其在不同年龄牦牛睾丸中的表达  

Cloning of Yak(Bos grunniens) AQP1 Gene and Its Expression in Yak Testis of Different Ages

作　　者：刘敏清 王亚营 何翃闳 潘阳阳 杨珊珊 高泽川 张同享 赵凌 王靖雷 崔燕 余四九 LIU Min-Qing;WANG Ya-Ying;HE Hong-Hong;PAN Yang-Yang;YANG Shan-Shan;GAO Ze-Chuan;ZHANG Tong-Xiang;ZHAO Ling;WANG Jing-Lei;CUI Yan;YU Si-Jiu(Technology and Research Center of Gansu Province for Embryonic Engineering of Bovine and Sheep&Goat,College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物医学院/甘肃省牛羊胚胎工程技术研究中心,兰州730070

出　　处：《农业生物技术学报》2021年第5期933-942,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31972760);甘肃省教育厅产业支撑引导项目(2019C-03);甘肃省教育厅创新能力提升项目(2019B-081)。

摘　　要：本研究旨在克隆牦牛(Bos grunniens)水通道蛋白1 (Aquaporin-1, AQP1)的基因序列及研究AQP1在不同年龄牦牛睾丸组织中的表达。采集初生(3 d)、青年(1岁)、成年(4岁)和老年(8岁)牦牛的睾丸样品,通过RT-PCR技术扩增得到AQP1基因的cDNA全长序列,并对其进行生物信息学分析。采用qRT-PCR、Western blot和免疫组织化学(immunohistochemistry)方法在基因和蛋白水平对其进行检测和定位。q RTPCR结果显示:随着牦牛年龄的增长,牦牛AQP1基因的表达水平呈先上升后下降的趋势,在青年睾丸中达到最高;在青年组中的表达量与其他组之间差异性均显著(P<0.05),而中年组与老年组差异不显著(P>0.05)。Western blot结果显示:随着牦牛年龄的增长,AQP1蛋白表达水平也呈现先上升后下降的趋势,在青年睾丸中达到最高;在青年组与其他组中的表达差异性显著(P<0.05),而初生组,中年组和老年组之间差异性均不显著(P>0.05)。免疫组织化学结果显示:AQP1蛋白在不同年龄牦牛睾丸中均有阳性表达,,主要表达在精子、精子细胞、各级生精细胞、睾丸间质细胞、支持细胞、管周肌样细胞和生精小管基膜中,各年龄表达位置基本一致。本实验结果表明,AQP1在青年组牦牛中的表达显著高于其他组(P<0.05),推测AQP1在青年牦牛睾丸中发挥着重要作用。本研究为进一步研究AQPs在牦牛睾丸中的作用机制提供理论依据。The aim of this study was to clone the gene sequence of Aquaporin-1(AQP1) in yak(Bos grunniens) and study the expression of AQP1 in yak testes of different ages. Testicular samples of newborn(3 day), young(1 year old), adult(4 years old) and old(8 years old) yak were collected. The full-length cDNA sequence of AQP1 gene was amplified by RT-PCR technology and analyzed by bioinformatics. qRT-PCR,Western blott and immunohistochemistry methods were used to detect and locate at the gene and protein level.The qRT-PCR results showed that as the yak grew older, the expression level of the yak AQP1 gene showed a trend of rising first and then falling, reaching the highest in the young testis;the expression level in the young group was significantly different from other groups(P<0.05), while the difference between the middle-aged group and the elderly group was not significant(P>0.05). Western blot results showed that as the yak grew older, the expression level of AQP1 protein also showed a trend of first rising and then falling, reaching the highest in the young testis;the expression difference between the young group and other groups was significant(P<0.05), while the differences between the newborn group, middle-aged group and old group were not significant(P>0.05). Immunohistochemical results showed that AQP1 protein is positively expressed in yak testes of different ages, and the expression sites are basically same, mainly expressed in sperm, sperm cells, spermatogenic cells, testicular stromal cells, supporting cells, and peritubular muscles like cells and seminiferous tubules in the basement membrane. The results of showed that the expression of AQP1 in the young yak group was significantly higher than that in the other groups(P<0.05). It is speculated that AQP1 plays an important role in the young yak testis. This study provides theoretical basis for further research on the mechanism of AQPs in the yak testis.

关 键 词：牦牛 睾丸 水通道蛋白1基因(AQP1) 克隆 

分 类 号：S857.29[农业科学—临床兽医学]