鸡Ash2l基因携带Flag标签的真核融合表达载体构建及表达分析  

Construction and Expression Analysis of Eukaryotic Fusion Expression Vector Carrying Flag Tag of Ash2l Gene in Chicken(Gallus gallus)

作　　者：张晨 左其生 邹艺琛 赵娟娟 张亚妮 李碧春 ZHANG Chen;ZUO Qi-Sheng;ZOUYi-Chen;ZHAO Juan-Juan;ZHANG Ya-Ni;LI Bi-Chun(Institutes of Agricultural Science and Technology Development/Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China/College of Animal Science and Technology,Yangzhou University Yangzhou,225009,China)

机构地区：[1]扬州大学农业科技发展研究院/教育部农业与农产品安全国际合作联合实验室/动物科学与技术学院,扬州225009

出　　处：《农业生物技术学报》2021年第6期1161-1168,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31572390,31872341);国际合作重点研发专项(2017YFE0108000);扬州市科技计划项目(YZ2019146)。

摘　　要：类ASH2蛋白(absent,small,or homeotic-like,ASH2L)是重要的组蛋白甲基化修饰酶,参与多种细胞发育过程。但其在雄性生殖细胞发生过程中的功能和分子机制尚不清晰。本研究旨在研究Ash2l在鸡(Gallus gallus)雄性生殖干细胞形成中的表达规律并构建Ash2l真核融合表达载体,利用NCBI保守结构域分析、UniPort、TMHMM Server v.2.0和SignalP在线网站分析ASH2L的保守结构域、亲疏水性、跨膜结构域和信号肽表达。收集18.5 d鸡(Gallus gallus)胚不同组织和不同阶段的鸡细胞胚胎干细胞(embryonic stem cells,ESC)、原始生殖细胞(primordial germ cell,PGC)、精原干细胞(spermatogonial stem cell,SSC)和鸡成纤维细胞(chicken embryonic fibroblast,CEF),qRT-PCR检测Ash2l的表达;双酶切和测序对重组载体pcDNA3.1-Ash2l-Flag进行鉴定;利用qRT-PCR和Western blot检测重组蛋白在PGC中的表达;通过免疫共沉淀(Co-immunoprecipitation,Co-IP)验证重组蛋白和互作蛋白的结合。结果显示,ASH2L蛋白含有2个保守结构域,SPRY(SPIa and ryanodine receptor,SPRY)-Ash2和PHD(plant homeodomain)-ash2p-like具有组蛋白甲基化酶特征结构域;其亲水性高、不含跨膜结构域且无信号肽表达。q RT-PCR检测结果发现,Ash2l在睾丸中表达最高,极显著高于其他组织(P<0.01),在PGC中表达极显著高于其他细胞(P<0.01)。双酶切结果表明,Ash2l正确插入pcDNA3.1载体,未发生移码和突变。qRT-PCR结果表明,PGC中重组载体可过量表达Ash2l基因;Western blot结果显示,PGC中成功表达重组蛋白。Co-IP结果发现,重组蛋白可与互作蛋白Dpy30组蛋白甲基化酶复合体调节亚基(Dpy-30 histone methyltransferase complex regulatory subunit,DPY30)和WD重复域5(WD repeat containing protein 5,WDR5)结合,形成复合体。本研究结果为探明Ash2l在鸡雄性生殖细胞形成中的功能和机制研究提供参考依据和技术手段。Absent,small,or homeotic-like(ASH2 L)is an important histone methylation modification enzyme involved in a variety of cell development.However,its function and molecular mechanism in the process of male germ cell development are not clear.The purpose of this study was to study the expression of Ash2 l in the formation of chicken(Gallus gallus)male reproductive stem cells and to construct Ash2 l eukaryotic fusion expression vector NCBI conserved domain analysis,UniPort,TMHMM Serverv.2.0 and SignalP online websites were used to analyze the conserved domain,hydrophilic and hydrophobic domain,transmembrane domain and signal peptide of ASH2 L.qRT-PCR was used to detect the expression of Ash2 l in different cells(embryonic stem cells(ESC),primordial germ cell(PGC)and spermatogonial stem cell(SSC))and tissues of 18.5 d chicken embryo.The recombinant vector pcDNA3.1-Ash2 l-Flag was identified by double restriction endonuclease digestion and sequencing.The expression of recombinant protein in PGC was detected by qRT-PCR and Western blot.The binding of recombinant protein and interaction protein was verified by Co-immunoprecipitation(Co-IP).The results showed that ASH2 L protein contained 2 conserved domains,SPIa and ryanodine receptor(SPRY)and plant homeodomain(PHD)had histone methylase characteristic domain,and had high hydrophilicity,no transmembrane domain and no signal peptide expression.The results of qRT-PCR showed that the expression of Ash2 l in testis was the highest,which was significantly higher than that in other tissues(P<0.01),and that in PGC was significantly higher than that in other cells(P<0.01).The results of double restriction endonuclease digestion showed that Ash2 l was correctly inserted into pcDNA3.1 vector without frameshift and mutation.qRT-PCR showed that the recombinant vector in PGC could overexpress Ash2 l gene,and Western blot indicated that the recombinant protein was successfully expressed in PGC.Co-IP results showed that the recombinant protein could bind to the interacting proteins Dpy-3

关 键 词：Ash2l 鸡 真核融合表达载体 雄性生殖干细胞 

分 类 号：S831.2[农业科学—畜牧学]