牦牛KDM2A基因克隆及其在不同组织和减数分裂进程的表达  

Cloning of Yak (Bos grunniens) KDM2A Gene and Its Expression in Different Tissues and Mitotic Progression

作　　者：杨显英[1] 熊显荣[1] 蔡雯祎 韩杰 阿果约达 李键[1] YANG Xian-Ying;XIONG Xian-Rong;CAI Wen-Yi;HAN Jie;A-GUO;Yue-Da;LI Jian(College of Life Science and Technology, Southwest Minzu University, Chengdu 610041, China)

机构地区：[1]西南民族大学生命科学与技术学院,成都610041

出　　处：《农业生物技术学报》2018年第7期1186-1194,共9页Journal of Agricultural Biotechnology

基　　金：四川省科技支撑计划(No.2017NZ0076);西南民族大学牦牛创新团队项目(No.13CXTD01);四川省肉牛现代繁殖技术研究与示范岗位项目

摘　　要：组蛋白去甲基化酶(histone demethylase,KDM)2A是细胞周期基因表达所必需的调控分子。为了研究牦牛(Bos grunniens)KDM2A基因的结构和功能,并进一步分析该基因在不同组织及卵母细胞减数分裂进程中的表达模式,本研究首先克隆获得牦牛KDM2A基因序列全长3 546 bp,包括完整的开放阅读框3 489 bp,编码氨基酸1 162个(Gen Bank登录号:MH345760)。牦牛KDM2A基因与其他物种的相似度与构建的系统进化树结果一致。牦牛KDM2A蛋白分析显示,其理论分子质量为132.7 k D,理论等电点为7.59,不稳定指数52.73,总平均亲水指数-0.587。KDM2A蛋白氨基酸序列中存在117个磷酸化位点,6个N-糖基化和2个O-糖基化位点;二级结构包括无规则卷曲、α螺旋和β折叠。KDM2A蛋白含有cupin超家族jumonji-C(Jmj C)结构域、锌指(CXXC zinc finger,ZF-CXXC)结构域、植物同源域(plant homeodomain,PHD)、F-盒蛋白(F-box,FBOX)结构域和有丝分裂相关蛋白(antagonist of mitotic exit network protein 1,AMN1)结构域,不含跨膜结构域和信号肽,主要在细胞核发挥生物学功能。KDM2A mRNA在子宫、脾脏和卵巢的表达量较高,极显著高于其他组织(P<0.01);KDM2A mRNA在卵母细胞减数分裂过程中均表达,其中MⅡ期的表达量极显著高于GⅤ期和MⅠ期(P<0.01),GⅤ期KDM2A的表达水平高于MⅠ期,但差异不显著(P>0.05)。本研究成功克隆了牦牛KDM2A基因序列,并明确其在组织和卵母细胞减数分裂进程中的表达模式,为深入探讨KDM2A基因在牦牛卵母细胞减数分裂进程中的作用机制提供了基础资料。Lysine demethylase 2A （KDM2A） is required for proper regulation of cell-cycle genes. The present study focused on the structure and function of KDM2A in yak （Bos grunniens）, and its expression pattern in meiosis process and different tissues. Total RNA of yak heart, liver, spleen, lung, kidney, gastric, small intestine, brain, ovary, uterus, and oocytes （G Ⅴ stage, M Ⅰ stage, and M Ⅱ stage） were extracted and reversely transcribed into cDNA. Specific primers were designed according to Bos indicus mRNA sequence of KAM2A gene. The coding sequences of KDM2A gene was cloned and analyzed by bioinformatics. The gene expression pattems in different tissues and oocytes （G Ⅴ stage, M Ⅰ stage, and M Ⅱ stage） were detected by quantitative real-time PCR （qRT-PCR）. The full-length sequence of yak KDM2A gene was 3 546 bp including a 3 489 bp open reading frame, encoding 1 162 amino acids （GenBank ID： MH345760）. The sequence alignment and phylogenetic tree of KDM2A compared yak to Bison bison bison, Capra hireus, Sus scrofa, Equus caballus, Homo sapiens, Felis catusg, Pongo abelii, Mus musculus and Gallus gallus was 99.8%, 98.5%, 95.9%, 95.3%, 95%, 94.5%, 92.8%, 91.4%, and 79.1%, respectively. There was a high homology among different species, and phyletic evolution was the same as their genetic relationship. The molecular weight of protein encoded by KDM2A gene in yak was 132.7 kD. The theory isoelectric point was 7.59, and the instability index was 52.73 and the grand average of hydroparthicity was -0.587. It was an unstable and hydrophilic protein. There were 117 phosphorylation sites, 6 N-glycosylation sites, and 20-glycosylations sites. The secondary structure of KDM2A consisted of β-overlap, u-helix and random coils. The amino acids sequence of KDM2A contains jumonji-C （JmjC）, CXXC zinc finger （ZF-CXXC）, plant homeodomain （PHD）, F-box （FBOX） and antagonist of mitotic exit network protein 1 （AMN1） domain. The protein was predicted to be lacking cross- membrane and

关 键 词：牦牛 组蛋白赖氨酸去甲基化酶2A基因(KDM2A1 卵母细胞 生物信息学 基因表达 

分 类 号：S823.85[农业科学—畜牧学]