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黏质沙雷氏菌灵菌红素合成基因簇异源表达及其潜在的温度调控机制

刘方晨 贾宪波 吴良泉 方宇 赵恪 林俊杰 陈龙军 张慧 林陈强 陈济琛

刘方晨,贾宪波,吴良泉,等. 黏质沙雷氏菌灵菌红素合成基因簇异源表达及其潜在的温度调控机制 [J]. 福建农业学报,2021,36(3):337−344 doi: 10.19303/j.issn.1008-0384.2021.03.013
引用本文: 刘方晨,贾宪波,吴良泉,等. 黏质沙雷氏菌灵菌红素合成基因簇异源表达及其潜在的温度调控机制 [J]. 福建农业学报,2021,36(3):337−344 doi: 10.19303/j.issn.1008-0384.2021.03.013
LIU F C, JIA X B, WU L Q, et al. Heterologous Expression and Temperature Regulation of Prodigiosin-synthesis Gene Cluster in Serratia marcecens [J]. Fujian Journal of Agricultural Sciences,2021,36(3):337−344 doi: 10.19303/j.issn.1008-0384.2021.03.013
Citation: LIU F C, JIA X B, WU L Q, et al. Heterologous Expression and Temperature Regulation of Prodigiosin-synthesis Gene Cluster in Serratia marcecens [J]. Fujian Journal of Agricultural Sciences,2021,36(3):337−344 doi: 10.19303/j.issn.1008-0384.2021.03.013

黏质沙雷氏菌灵菌红素合成基因簇异源表达及其潜在的温度调控机制

doi: 10.19303/j.issn.1008-0384.2021.03.013
基金项目: 国家自然科学基金青年基金项目(31800068);福建省农业科学院国家自然科学基金项目延伸研究专项计划(AGY2018-1);福建省科技厅区域发展项目(2019Y3005)
详细信息
    作者简介:

    刘方晨(1996−)男,硕士,研究方向:环境微生物(E-mail:liufc1996@163.com

    通讯作者:

    贾宪波(1986−)男,博士,助理研究员,研究方向:环境微生物(E-mail:xbj2011@163.com

  • 中图分类号: S 182

Heterologous Expression and Temperature Regulation of Prodigiosin-synthesis Gene Cluster in Serratia marcecens

  • 摘要:   目的  构建黏质沙雷氏菌FZSF02灵菌红素合成基因簇的异源表达菌株,探究其合成灵菌红素可能的温度调控机制。  方法  构建3种携带不同启动子的异源表达大肠杆菌菌株,并利用实时荧光定量PCR(RT-qPCR)检测FZSF02中pig基因簇pigApigFpigN基因在37 ℃与28 ℃培养条件下的转录水平差异。  结果  在28 ℃低温条件下,3种不同的启动子均能在大肠杆菌中启动各自重组质粒所携带的pig基因簇表达并合成灵菌红素,其中携带T7强启动子的重组菌株经IPTG诱导后可以在37 ℃少量合成色素,其余菌株37 ℃不能合成色素。RT-qPCR结果显示在黏质沙雷氏菌中上述基因转录水平在37 ℃条件下较28 ℃出现下调。  结论  黏质沙雷氏菌FZSF02之所以能够在28 ℃合成灵菌红素而在37 ℃无法合成灵菌红素,一方面由于pig基因簇的编码基因转录水平下调,另一方面是由基因簇编码的合成灵菌红素所需前体MBC和MAP的一种或者几种关键酶在相对较高的温度(37 ℃)条件下活性受到抑制导致。
  • 图  1  pig基因簇、pig基因簇和pig启动子序列以及3种重组质粒所连片段的验证PCR扩增结果

    注:M:DNA maker。A:1,pig基因簇;2,pig基因簇启动子与pig基因簇。B:(1,2)M13引物PCR扩增产物;3,T7引物PCR扩增产物。

    Figure  1.  PCR amplification results on pig gene cluster, pig gene cluster and pig promoter, and validation on sequences of 3 recombinant plasmid segments

    Note:M: DNA maker. A: pig gene cluster (1); pig gene cluster and pig promoter (2). B: M13 primer PCR amplification product (1 and 2); T7 primer PCR amplification product (3).

    图  2  3种重组质粒的构建过程

    注:A: pTOPO-Blunt Simple-ppPig重组质粒;B: pEASY®-Blunt E2-Pig重组质粒;C: pTOPO-Blunt Simple-kpPig重组质粒。

    Figure  2.  Construction of 3 recombinant plasmids

    Note:A: pTOPO-Blunt Simple-ppPig recombinant plasmid. B: pEASY®-Blunt E2-Pig recombinant plasmid. C: pTOPO-Blunt Simple-kpPig recombinant plasmid.

    图  3  3种重组菌株在28 ℃和37 ℃培养条件下灵菌红素的合成情况

    注:A:28 ℃培养pTOPO-ppPig重组菌株;B:28 ℃培养pTOPO-kpPig重组菌株;C:28 ℃培养pEASY-pig重组菌株;D:37 ℃培养pTOPO-ppPig重组菌株;E:37 ℃培养pTOPO-kpPig重组菌株;F:37 ℃培养pEASY-pig重组株。

    Figure  3.  Prodigiosin synthesis of 3 recombinant strains cultured at 28 ℃ and 37 ℃

    Note: A: pTOPO-ppPig recombinant strains at 28 ℃. B: pTOPO-kpPig recombinant strains at 28 ℃. C: pEASY-pig recombinant strains at 28 ℃. D: pTOPO-ppPig recombinant strains at 37 ℃. E: pTOPO-kpPig recombinant strains at 37 ℃. F: pEASY-pig recombinant strains at 37 ℃.

    图  4  粘质沙雷氏菌在不同温度条件下所选基因的转录水平倍数变化

    Figure  4.  Expressions of selected genes of S. marcecens at different temperatures

    表  1  供试菌株和质粒

    Table  1.   Bacterial strains and plasmids used in this study

    细菌 Bacterial strains菌株 Strain参考/来源 Reference/source
    Serratia marcescens FZSF02(WT) This work(本实验)
    E.coli DH5α This work(本实验)
    BL21(DE3) This work(本实验)
    质粒 Plasmids 注释 Note 参考/来源 Reference/source
    pTOPO CV22-Zero Background pTOPO-Blunt Simple Cloning Kit Purchased from Aidlab(购自艾伯莱生物公司)
    pEASY pEASY®- Blunt E2 Expression Kit Purchase from TransGen Biotech
    (购自全式金生物公司)
    pTOPO-kan Used for the amplification of universal promoter and Kan resistance genes This work(本实验)
    pTOPO-ppPig A pTOPO based plasmid containing the pig cluster and pig promoter This work(本实验)
    pEASY-Pig A pEASY based plasmid containing the pig cluster This work(本实验)
    pTOPO-kpPig A pTOPO based plasmid containing the pig cluster and Kan promoter This work(本实验)
    下载: 导出CSV

    表  2  供试引物

    Table  2.   Primers used in this study

    引物 Primers序列 Sequences(5′→3′)用途 purpose
    ExpF ATGGATTTTAACTTATCAAGCGA Clone a pig gene cluster(克隆pig基簇)
    ExpR TTACAGCACGAAAGGAATGAAACA
    CluspigF TTTTTCCTCCGGAATGCTCCTGC Clone pig gene clusters and pig promoter
    (克隆pig基因簇及其启动子)
    CluspigR TTACAGCACGAAAGGAATGAAAC
    KanproF2 ATTCCTTTCGTGCTGTAATCAGAA
    TTGGTTAATTGGTTGTA Linearize the attached pTOPO
    KanproR2 TGATAAGTTAAAATCCATAACACC vector and Kan promoter
    (线性化pTOPO载体和Kan启动子)
    CCTTGTATTACTGTTA
    M13F TGTAAACGACGGCCAGT Check the size of the gene attached to the pTOPO vector
    (验证pTOPO载体所连接的基因大小)
    M13R CAGCAAACAGCTATGACC
    16SF CGTTACTCGCAGAAGAAGCA Used for qPCR amplification(用于qPCR扩增)
    16SR TCACCGCTACACCTGGAA
    PigAF CGCCATCTTCCACGATTCAA Used for qPCR amplification(用于qPCR扩增)
    PigAR CATTAGCCGACACTGTTCCA
    PigFF CACGGTATTCGGCGATGAC Used for qPCR amplification(用于qPCR扩增)
    PigFR CACGGTGTTGCGAGAAGT
    PigNF CGGTTACCCTGGTCTATTG Used for qPCR amplification(用于qPCR扩增)
    PigNR TGTCAGCACGATGTTCAT
    下载: 导出CSV

    表  3  菌株在535 nm下的吸光度

    Table  3.   Absorbance at 535 nm of recombinant strains

    菌株 strains吸光度 Absorbance
    28 ℃37 ℃
    DH5α(pTOPO-ppPig) 0.435 0.002
    DH5α(pTOPO-kpPig) 0.468 0.001
    DH5α(pEASY-Pig) 0.394 0.026
    FZSF02 1.056 0.001
    下载: 导出CSV
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出版历程
  • 收稿日期:  2020-11-24
  • 修回日期:  2021-01-28
  • 网络出版日期:  2021-03-27
  • 刊出日期:  2021-03-31

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