鸡(Gallus gallus)中介导ANP32家族蛋白细胞核定位的关键氨基酸位点的鉴定  

Identification of Key Amino Acids Determining the Nuclear Localization of Chicken(Gallus gallus)ANP32 Family Proteins

作　　者：邢静如 石海英 崔平 张前勇 段志强 XING Jingru;SHI Haiying;CUI Ping;ZHANG Qianyong;DUAN Zhiqiang(Key Laboratory of Ministry of Education for Animal Genetics,Breeding and Reproduction in the Plateau Mountains Region,Key Laboratory of Animal Genetics,Breeding and Reproduction in Guizhou,College of Animal Sciences,Guizhou University,Guiyang,550025;Management and Service Center of Animal Husbandry and Veterinary of Binzhou,Binzhou,256600)

机构地区：[1]贵州大学动物科学学院,高原山地动物遗传育种与繁殖教育部重点实验室,贵州省动物遗传育种与繁殖重点实验室,贵阳550025 [2]滨州市畜牧兽医管理服务中心,滨州256600

出　　处：《基因组学与应用生物学》2023年第10期1088-1099,共12页Genomics and Applied Biology

基　　金：国家自然科学基金项目(31960698,31760732);贵州省科学技术基金项目(黔科合基础[2020]1Y134号)共同资助。

摘　　要：本研究旨在鉴定鸡(Gallus gallus)中介导ANP32家族蛋白核定位信号(nuclear localization signal, NLS)中的关键碱性氨基酸位点。通过生物信息学软件对鸡ANP32家族蛋白二级结构、三级结构、氨基酸同源性、结构域保守性和NLS(KRKR)保守性进行分析;同时,构建表达鸡ANP32家族蛋白NLS及其碱性氨基酸位点突变体的重组真核表达载体并转染HEK-293T细胞,通过观察分析其亚细胞定位来鉴定NLS中的关键碱性氨基酸位点。生物信息学分析结果显示,鸡ANP32家族蛋白均以α-螺旋和无规则卷曲为主;其与鸭ANP32家族蛋白的氨基酸同源性最高且达到98.8%;其与鸭ANP32家族蛋白功能结构域保守性最高,LRR和LCAR结构域完全保守;并且鸡ANP32家族蛋白NLS在不同物种间高度保守,NLS均为KRKR。亚细胞定位分析发现,鸡ANP32家族蛋白及其NLS的融合蛋白均主要定位于细胞核,而NLS中的第一个K、第一个R和第二个K突变后导致融合蛋白出现在细胞质,可以推断它们是鸡中介导ANP32家族蛋白细胞核定位的关键氨基酸位点。本研究发现,鸡ANP32家族蛋白NLS具有与全长蛋白相同的细胞核定位特征,且在不同物种间具有高度保守性。鸡ANP32家族蛋白细胞核定位的关键氨基酸位点的鉴定为后续研究其入核转运机制奠定了理论基础。The purpose of this study was to identify the key basic amino acid residues in the nuclear localization signal(NLS)that mediate the nuclear localization of chicken(Gallus gallus)ANP32 family proteins.The secondary structure,tertiary structure,amino acid homology,domain conservation and NLS(KRKR)conservation of chicken ANP32 family proteins were analyzed by using bioinformatic softwares.Meanwhile,the recombinant eukaryotic expression vectors expressing the NLS of chicken ANP32 family proteins and its basic amino acid mutants were constructed and then transfected into HEK-293T cells.The key basic amino acid sites in NLS were identified by observing and analyzing their subcellular localization.The results of bioinformatic analysis showed that chicken ANP32 family proteins were mainly composed ofα-helix and random coil;It had the highest amino acid homology with duck ANP32 family proteins and reached 98.8%;It had the most conserved functional domain compared with duck ANP32 family proteins,LRR and LCAR domains were completely conserved.Meanwhile,the chicken ANP32 family proteins NLS was highly conserved among different species,and the NLS was KRKR.Subcellular localization analysis showed that the chicken ANP32 family proteins and their NLS fusion proteins were mainly located in the nucleus.Mutation of the first K,the first R,and the second K in NLS caused the cytoplasmic distribution of fusion proteins,thus concluding that the three residues were the key amino acid sites mediating the nuclear localization of chicken ANP32 family proteins.This study found that the chicken ANP32 family proteins NLS has the same nuclear localization characteristics as the full-length protein,and is highly conserved among different species.The identification of the key amino acid sites of the nuclear localization of chicken ANP32 family proteins provides a theoretical basis for the subsequent study of the mechanism of the nuclear transport of chicken ANP32 family proteins.

关 键 词：鸡(Gallus gallus) ANP32 亚细胞定位 核定位信号 

分 类 号：S831[农业科学—畜牧学]