小鼠RNA结合蛋白QKI-5的生物信息学和组织表达分析及其过表达对癌症相关基因表达的影响  

Bioinformatics and Tissue Expression Analysis of Mouse RNA Binding Protein QKI-5 and Its Overexpression Effect on Cancer-Related Genes Expression

作　　者：王逢博 高登科 李超[1,2] 李丹 刘薇 张海森 杨王浩 靳亚平 陈华涛[1,2] WANG Fengbo;GAO Dengke;LI Chao;LI Dan;LIU Wei;ZHANG Haisen;YANG Wanghao;JIN Yaping;CHEN Huatao(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi 712100,China;Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural Affairs,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]农业农村部动物生物技术重点实验室,陕西杨凌712100

出　　处：《江西农业大学学报》2023年第2期453-466,共14页Acta Agriculturae Universitatis Jiangxiensis

基　　金：国家自然科学基金项目(31771301)。

摘　　要：【目的】旨在构建小鼠Quaking基因的主要转录本Quaking-5(Qki-5)的真核表达载体,利用生物信息学软件预测分析其结构与功能特征,检测该基因的组织表达谱,并在小鼠AML12肝细胞系中探究Qki-5过表达对癌症相关基因表达的影响。【方法】提取小鼠肝脏组织的总RNA,反转录为cDNA,通过PCR扩增Qki-5转录本的蛋白编码区(Coding Sequence,CDS)全长,将获得的目的片段与线性化载体pcDNA3.1-Puro-N-3HA进行同源重组连接以获得pcDNA3.1-mQki-5。通过在线软件对Qki-5基因序列及其编码蛋白进行生物信息学分析,并利用实时荧光定量PCR(qRT-PCR)检测Qki-5基因在小鼠不同组织中的表达谱,同时使用免疫组织化学技术检测QKI-5蛋白在小鼠肝脏组织的表达分布。之后,将pcDNA3.1-mQki-5重组质粒和pcDNA3.1-Puro-N-3HA空质粒分别转染至AML12细胞,通过qPCR和Western Blotting(WB)检测Qki-5基因在mRNA和蛋白水平的表达,并进一步检测Qki-5基因在AML12细胞中过表达后对癌症相关基因表达的影响。【结果】成功构建了pcDNA3.1-mQki-5真核表达载体;小鼠Qki-5基因的CDS区长度为1026 bp,共编码341个氨基酸;小鼠QKI-5蛋白为亲水性蛋白,不含跨膜结构和信号肽,存在26个磷酸化位点,且小鼠QKI-5蛋白的三级结构与人和大鼠完全一致;在检测的12个组织中,Qki-5在小鼠全脑的表达量最高,在十二指肠的表达量最低;QKI-5在小鼠肝脏组织中主要表达于肝细胞细胞核中;在AML12细胞过表达QKI-5后,会引起癌症相关基因P53和Ccnd1的mRNA表达水平显著升高。【结论】研究成功构建了小鼠Qki-5基因真核表达载体;Qki-5基因在小鼠全脑、肺脏等组织中表达量较高;QKI-5主要表达分布于小鼠肝细胞细胞核内;此外,在AML12细胞中QKI-5的过表达会引起P53和Ccnd1的表达水平升高。研究为进一步探究小鼠Qki-5基因的功能提供了关键材料。[Objective]This study was designed to construct the eukaryotic expression vector of Quaking-5(Qki-5,the main transcript of mouse Quaking gene).Its structure and functional characteristics were predicted and analyzed by bioinformatics software,the tissue expression profiles of Qki-5 gene was detected,and its overexpression effect on the expression of cancer-related genes in mouse AML12 hepatocyte cell line were explored.[Method]The total RNA was extracted from mouse liver tissue,and then it was reversely transcribed into cDNA.The full-length of mouse Qki-5 protein coding sequence(CDS)was amplified by PCR.The target fragment was ligated with the linearized vector pcDNA3.1-Puro-N-3HA to obtain pcDNA3.1-mQki-5 with homologous recombination.The Qki-5 gene sequence and its encoded protein was analyzed by bioinformatics online software.The expression profiles of Qki-5 gene in different tissues of mouse were detected by real-time fluorescence quantitative PCR(qRT-PCR),and the expression distribution of QKI-5 protein in mouse liver was detected by immunohistochemical staining.After that,pcDNA3.1-mQki-5 recombinant plasmid and pcDNA3.1-Puro-N-3HA empty plasmid were transfected into AML12 cells respectively.The expression of Qki-5 gene at mRNA and protein levels were detected by qPCR and Western Blotting(WB),and its overexpression effect on the expression of cancer-related genes was further examined in AML12 cells.[Result]The pcDNA3.1-mQki-5 eukaryotic expression vector was successfully constructed.The CDS region of mouse Qki-5 gene is 1026 bp in length,encoding 341 amino acids.Mouse QKI-5 protein is a hydrophilic protein without transmembrane structure and signal peptide,and it has 26 phosphorylation sites.The tertiary structure of mouse QKI-5 protein is the same with that of human and rat.Among the twelve tissues,the expression level of Qki-5 was the highest in the whole brain and the lowest in the duodenum of mouse.QKI-5 was mainly expressed in mouse hepatocytes nucleus.The mRNA levels of cancer-related genes P53 and Ccn

关 键 词：RNA结合蛋白 QKI 表达谱 哺乳动物 癌症 

分 类 号：S852.2[农业科学—基础兽医学]