| 摘要: |
| 【目的】对鸡色氨酸天冬氨酸重复域5（WD repeat domain 5，WDR5）进行生物信息学分析，构建真核表达载体，并检测其对原始生殖细胞（Primordial germ cells，PGCs）形成相关基因表达的影响，探索WDR5在鸡PGCs形成中的调控作用。【方法】利用ProtParam、 NCBI保守结构域分析、Uniport、TMHMM Server v.2.0和SignalP在线网站，分析WDR5的氨基酸序列、保守结构域、亲疏水性、跨膜结构域和信号肽表达。利用SWISS-MODEL进行同源建模预测其三级结构，借助String网站进行互作蛋白的预测。构建重组载体pcDNA3.1-WDR5-GST，进行双酶切和测序鉴定；将重组载体pcDNA3.1-WDR5-GST转染PGCs，采用实时荧光定量PCR（RT-qPCR）和Western blot检验WDR5重组蛋白在PGC中的表达情况；用RT-qPCR检测过表达WDR5对PGCs形成相关基因(DDX4、C-KIT、BLIMP1、LIN28B和PRDM14)表达水平的影响。【结果】WDR5蛋白含有1个高度保守的WD40结构域，内含7个典型的WD40重复序列；其亲水性高、不含跨膜结构域、无信号肽表达，可用于构建重组真核表达载体。同源建模和互作蛋白预测显示WDR5高度保守，互作蛋白为类ASH2蛋白（ASH2 Like，ASH2L）、RB结合蛋白5（RB Binding Protein 5，RBBP5）、DPY30和KAT8调控因子NSL复合物亚基3（KAT8 Regulatory NSL Complex Subunit 3，KANSL3）。双酶切和测序结果表明，重组载体pcDNA3.1-WDR5-GST构建成功。RT-qPCR结果显示PGC中重组载体可使WDR5过量表达;Western blot结果表明，PGC中可表达重组蛋白。WDR5过表达可使PGCs形成相关基因的表达水平升高。【结论】构建的WDR5重组载体在鸡PGCs中能够表达；WDR5基因表达上调后，PGCs形成相关基因表达水平升高，推测其可调控鸡PGCs的形成。 |
| 关键词: WDR5 鸡 真核融合表达载体 原始生殖干细胞 |
| DOI： |
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| 基金项目:国家自然科学基金项目（31772582，31972547）；国际合作重点研发专项(2017YFE0108000)；扬州市科技计划项目（YZ2019146） |
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| Construction of eukaryotic fusion expression vector of chicken (Gallus gallus) WDR5 gene and its effect on expression of genes related to PGCs formation |
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ZHANG Chen,ZUO Qisheng,ZOU Yichen,et al
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| Abstract: |
| 【Objective】This study analyzed the chicken WD repeat domain 5 (WDR5) by bioinformatics,constructed a eukaryotic fusion expression vector of WDR5,detected primordial germ cells (PGCs) related genes,and explored the regulatory mechanism of WDR5 in PGCs formation.【Method】The ProtParam,NCBI conserved domain analysis,Uniport,TMHMM Server v.2.0 and SignalP online website were used to analyze the amino acid sequence,conserved domain,hydrophobicity,transmembrane domain and signal peptide expression of WDR5.SWISS-MODEL was used for homology modeling to predict its tertiary structure,and String website was used to predict interacting proteins.The recombinant vector pcDNA3.1-WDR5-GST was identified by double enzyme digestion and sequencing,and the expression of WDR5 in PGC was verified by RT-qPCR and Western blot.The RT-qPCR was used to detect expression of PGCs related genes (DDX4,C-KIT,BLIMP1,LIN28B and PRDM14) after overexpression of WDR5.【Result】The WDR5 protein contained a highly conserved structural domain WD40 domain with 7 typical WD40 repeats.WDR5 was highly hydrophilic without transmembrane domain and signal peptide expression,and it can be used for constructing recombinant eukaryotic expression vector.Homology modeling and prediction of interacting proteins showed that WDR5 was highly conserved,and the interacting proteins included ASH2 Like (ASH2L),RB Binding Protein 5 (RBBP5),DPY30 and KAT8 Regulatory NSL Complex Subunit 3 (KANSL3).Double enzyme digestion and sequencing showed that the recombinant vector pcdna3.1-wdr5 gst was successfully constructed.The results of RT-qPCR showed that the recombinant vector in PGC could overexpress WDR5.The results of Western blot showed that the recombinant protein could be expressed in PGC.The expression of PGCs related genes increased with overexpression of WDR5.【Conclusion】WDR5 recombinant vector could express in chicken PGCs.PGCs formation related genes increased after overexpression of WDR5,indicating that WDR5 could regulate PGCs formation. |
| Key words: WDR5 chicken eukaryotic fusion expression vector primordial germ cell |
