关中奶山羊Prm1基因的克隆及真核表达  

Cloning of Dairy Goat Prm1 Gene CDS and Construction of the Eukaryotic Expression Vector

作　　者：李明昭[1] 刘超[1] 孙军伟[1] 朱海鲸[1] 曹晖[1] 吕晓[1] 仇普斌[1] 华进联[1] 

机构地区：[1]西北农林科技大学动物医学院/陕西省干细胞工程技术研究中心/农业部动物生物技术重点开放性实验室,陕西杨凌712100

出　　处：《中国农业科学》2012年第6期1183-1190,共8页Scientia Agricultura Sinica

基　　金：国家自然科学基金(30200137);教育部重点科研项目(109148);中国博士后基金(20080431253);中国博士后科学基金特别资助项目(200801438);留学回国人员科研启动费(14110101);陕西省科技攻关(2008K02-05);西北农林科技大学青年学术骨干支持计划(01140301)

摘　　要：【目的】研究雄性奶山羊Prm1基因的表达规律并克隆该基因,为奶山羊精子发生过程的研究提供分子基础。【方法】利用PCR技术从关中奶山羊睾丸组织中扩增出Prm1基因的全CDS序列,对其进行同源性比较和进化比较。同时用PCR、western blotting及免疫组化技术检测Prm1在不同年龄雄性关中奶山羊睾丸中的表达。将克隆出的奶山羊Prm1基因与pIRES-GFP载体连接构建重组真核表达载体,转染GC1细胞和人的脐带基质细胞检测Prm1基因的超表达情况。【结果】Prm1基因在成年奶山羊睾丸组织中的表达量很高,且只定位于精子头部,在其它时期的睾丸组织中基本不表达。同时扩增得到了关中奶山羊Prm1基因CDS序列,与其它物种比对,发现其与许多物种具有较高的同源性,与牛的同源性高达97.4%,最后在GC1细胞和人的脐带基质细胞中进行了超表达。【结论】得到了关中奶山羊Prm1基因的表达规律及完整的CDS序列。【objective】The objective of the experiment is to study the Prm1 gene of dairy goat,and for further research on dairy goat spermatogenesis.【Method】The CDS of Prm1 gene was cloned by PCR from Guanzhong dairy goat testis tissue.The obtained sequence was constructed into the eukaryotic expression vector pIRES-GFP,and then transfected to GC1 cell line and human umbilical cord cells.PCR and immunohistochemistry were used to test the expression of Prm1 at different ages of dairy goat testis.【Result】In dairy goats,the expression of Prm1 gene was high and specific in adult testis,located only in the head of sperm.The CDS of Prm1 gene of Guanzhong dairy goat was amplified and compared with the sequences of other species.The results showed that they shared a high homology,being 97.4% between dairy goats and bovine.The Prm1 gene was overexpressed in GC1 cells and human umbilical cord cells to confirm its function.【Conclusion】 The complete CDS of dairy goat Prm1 gene and its expression patterns were obtained.

关 键 词：关中奶山羊 Prm1基因 基因克隆 表达规律 

分 类 号：S827[农业科学—畜牧学]