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  • ISSN 1006-3080
  • CN 31-1691/TQ

一种新型的曲霉属洁净可逆性诱导表达系统

李宝意 周胜敏

李宝意, 周胜敏. 一种新型的曲霉属洁净可逆性诱导表达系统[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20220407001
引用本文: 李宝意, 周胜敏. 一种新型的曲霉属洁净可逆性诱导表达系统[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20220407001
Li Baoyi, Zhou Shengmin. Construction and Optimization of a Novel Expression System Using H2O2 as Inducer in Aspergillus[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.20220407001
Citation: Li Baoyi, Zhou Shengmin. Construction and Optimization of a Novel Expression System Using H2O2 as Inducer in Aspergillus[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.20220407001

一种新型的曲霉属洁净可逆性诱导表达系统

doi: 10.14135/j.cnki.1006-3080.20220407001
基金项目: 基金项目类别(项目编号) 资助项目
详细信息
    作者简介:

    李宝意(1991—),女,安徽芜湖,硕士,主要研究方向:基础生物学、合成生物学、化学生物学。E-mail:476089724@qq.com

    通讯作者:

    周胜敏, E-mail:zhoushengmin@ecust.edu.cm

  • 中图分类号: Q78

Construction and Optimization of a Novel Expression System Using H2O2 as Inducer in Aspergillus

  • 摘要: 构建一种以构巢曲霉为表达宿主,以AnPrxA启动子为表达用启动子,以过氧化氢为表达诱导剂的新型高效的曲霉表达体系。评估构巢曲霉过氧化物氧还酶编码基因(AnPrxA)含启动子(PPRX)的上游调控区在双氧水诱导和非诱导条件下介导绿色荧光蛋白(GFP)表达的特征。构建4个含不同长度启动子(PPRX)表达盒的克隆载体并转化尿嘧啶生物合成缺陷型构巢曲霉;分析AnPrxA基因上游2033 bp序列(−2033~−1)中潜在的转录调控元件;荧光定量PCR测定表达盒拷贝数;考察与PPRX融合的GFP表达情况;探索双氧水诱导浓度和作用时间。结果表明,AnPrxA基因翻译起始密码子上游2033 bp序列至少包含9种真菌中常见的基因表达调控元件,其中大多数呈多拷贝排列。虽然上游2033 bp的区域对gfp的转录均有贡献,但就GFP蛋白表达而言,AnPrxA上游500 bp的区域已足够。在0.5-2.0 mmol/L的最佳双氧水诱导条件下,PPRX介导GFP表达的最大诱导比率为3倍,与先前鉴定的两种曲霉属双氧水诱导型启动子相似,但目标蛋白的相对表达强度高于两者。鉴于PPRX介导gfp转录的诱导比率(30倍)显著高于其蛋白表达的诱导比率(3倍),推测目的基因5’端编码区的部分序列可能影响蛋白质的翻译。

     

  • 图  1  过氧化物氧还酶基因启动子介导的绿色荧光蛋白报告基因表达系统的构建

    Figure  1.  Construction of GFP reporter gene expression system mediated by the peroxiredoxin gene promoter

    图  2  pPPRX-GFP系列重组质粒的酶切鉴定。

    Figure  2.  Identification of recombinant plasmids of the pPPRX-GFPs by digestion

    图  3  过氧化物氧还酶基因起始密码子上游序列中的潜在调控元件。

    Figure  3.  Potential regulatory elements in the sequence of the PRX promoter of the A. nidulans

    图  4  绿色荧光蛋白报告基因表达盒整合拷贝与表达量的对应关系

    Figure  4.  Correspondence between GFP copy number and Specific fluorescence

    图  5  过氧化物氧还酶基因4种长度的启动子序列在诱导和非诱导条件下介导绿色荧光蛋白报告基因表达的特征

    Figure  5.  Characteristics of four lengths of promoter sequences of PRX mediating GFP expression under inducible and non-inducible conditions

    图  6  过氧化物氧还酶基因启动子序列在双氧水诱导和非诱导条件下介导绿色荧光蛋白报告基因表达的比较

    Figure  6.  Comparison of PRX promoter sequences mediating green fluorescent protein reporter gene expression under H2O2 induced and non-induced conditions

    图  7  双氧水不同浓度对绿色荧光蛋白报告基因表达的诱导特性。

    Figure  7.  Effect of GFP expression under different concentrations of H2O2

    表  1  本文所用的PCR扩增引物

    Table  1.   Primers used in this study

    PrimerPrimer base sequences(5’Š3’)
    FpyrGATTCCCCGCGGGAATTCGATACCTGTCG
    RpyrGGCAATCCGCGGTCAGTGCTTGTCTACCAG
    P1-prx2033TTTTGCCCTTGCTGCCTC
    P2-prx2033CGGGGGATCCACTAGTGGTTACGACTTCTGGAAAAC
    P1-prx1500CAGGGGCTCGAAAACTG
    P2-prx1500CGGGGGATCCACTAGTATAGATAGTATTCCAGGTTGATTTCG
    P1-prx1000AACACGGATGCCAACTGC
    P2-prx1000CGGGGGATCCACTAGTCTATTAAGGATACGTCTGGC
    P1-prx500CGTCGCTCGAAGTAGCAG
    P2-prx500CGGGGGATCCACTAGTCGGCGATGTGACAAACAG
    P3-prxGTTCTTCTCCTTTACTCATTGTGTAAGATAGTGGTTG
    P4-prxGGATGAACTATACAAATAATAGTGATTTAATAGCTCC
    P5-prxACCGCGGTGGCGGCCGCAAAGAAGGATTACCTCTAAAC
    P6-prxTTCGAGATCAGGGGAGG
    FgfpATGAGTAAAGGAGAAGAAC
    RgfpTTATTTGTATAGTTCATCCATGCC
    F-YG-GFPCTGTCCACACAATCTGCCCT
    R-YG-GFPTGCCATGTGTAATCCCAGCA
    F-YG-ACTAAGCCCCCATCAATCCCAAG
    R-YG-ACTTAGAGACGTAGAAGGCGGGA
    下载: 导出CSV

    表  2  方差分析表

    Table  2.   ANOVA Table

    SSdfMSFP
    Groups172.0035506357.334516864.0495359630.050468081
    Error113.2663444814.15829304
    Total285.26989491125.93362681
    下载: 导出CSV
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  • 收稿日期:  2022-04-07
  • 网络出版日期:  2022-09-06

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