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普城沙雷氏菌ACCC 02146产灵菌红素调控基因的鉴定

丁霞飞 贾宪波 林陈强 庄振宏 陈济琛

丁霞飞,贾宪波,林陈强,等. 普城沙雷氏菌ACCC 02146产灵菌红素调控基因的鉴定 [J]. 福建农业学报,2023,38(4):485−496 doi: 10.19303/j.issn.1008-0384.2023.04.013
引用本文: 丁霞飞,贾宪波,林陈强,等. 普城沙雷氏菌ACCC 02146产灵菌红素调控基因的鉴定 [J]. 福建农业学报,2023,38(4):485−496 doi: 10.19303/j.issn.1008-0384.2023.04.013
DING X F, JIA X B, LIN C Q, et al. Prodigiosin-producing Genes in Serratia plymuthica ACCC 02146 [J]. Fujian Journal of Agricultural Sciences,2023,38(4):485−496 doi: 10.19303/j.issn.1008-0384.2023.04.013
Citation: DING X F, JIA X B, LIN C Q, et al. Prodigiosin-producing Genes in Serratia plymuthica ACCC 02146 [J]. Fujian Journal of Agricultural Sciences,2023,38(4):485−496 doi: 10.19303/j.issn.1008-0384.2023.04.013

普城沙雷氏菌ACCC 02146产灵菌红素调控基因的鉴定

doi: 10.19303/j.issn.1008-0384.2023.04.013
基金项目: 福建省科技计划公益类专项(2020R1025002);福建省农业科学院科技创新团队建设项目(CXTD2021002-3)
详细信息
    作者简介:

    丁霞飞(1995−),女,硕士,研究方向:生物化学与分子生物学(E-mail:dxf925@163.com

    通讯作者:

    陈济琛(1964−),男,研究员,研究方向:农业微生物(E-mail:chenjichen2001@163.com

  • 中图分类号: Q81

Prodigiosin-producing Genes in Serratia plymuthica ACCC 02146

  • 摘要:   目的  鉴定影响普城沙雷氏菌ACCC 02146产灵菌红素能力的基因,构建ACCC 02146的转座子突变体文库,为进一步研究普城沙雷氏菌ACCC 02146灵菌红素合成机制奠定基础。  方法  采用平板划线法从菌种保藏中心购买菌株和实验室保藏菌株中获得15株产灵菌红素的菌株。采用16S rRNA基因测序法鉴定各菌株,邻接建树将其分类,并比较不同类别菌株灵菌红素合成基因簇启动子序列差异,观察各菌株产色能力。对16S rDNA序列和灵菌红素合成基因簇启动子序列均有异于其他菌株的普城沙雷氏菌ACCC 02146合成灵菌红素的调控基因展开研究。构建ACCC 02146的转座子突变体文库,筛选出产色能力明显改变的克隆子并鉴定出对应的转座子插入突变基因。  结果  该突变体文库中有74个突变体表现出产灵菌红素能力的变化。其中25个突变体转座子插入发生在pigApigBpigCpigDpigH 等5个灵菌红素合成簇基因上,49个突变体转座子插入基因为灵菌红素合成基因簇之外的基因。在鉴定到的产色能力改变的突变体中,麦芽糖O-乙酰基转移酶基因突变菌株有6个,二氢乳清酸脱氢酶基因突变菌株有4个,MarR家族转录因子SlyA基因突变体有3个,winged helix家族的双组分转录调控因子RstA基因突变体有3个,H-醌氧化还原酶亚基I基因突变菌株有3个,NADH-醌氧化还原酶G链基因突变菌株有3个,肽基脯氨酰异构酶B基因突变菌株有3个,其他突变基因对应的克隆子数量为1~2个。  结论  在沙雷氏菌中,除了灵菌红素合成簇基因调控灵菌红素合成,推测灵菌红素合成簇外编码相关酶、转录调控因子和一些结构蛋白的基因通过直接或间接途径在不同程度上调控了灵菌红素的合成。
  • 图  1  菌株系统发育树

    Figure  1.  Phylogenetic tree of Serratia sp.

    图  2  各菌株灵菌红素合成能力比较

    Figure  2.  Prodigiosin synthesis capabilities of Serratia sp.

    图  3  3种不同转录调控因子突变体菌落

    a:突变体F4-4;b:突变体B4-1;c:突变体H8-1。

    Figure  3.  Colonies of 3 type transcriptional regulatory factor mutants

    a: Mutant F4-4; b: Mutant B4-1; c: Mutant H8-1.

    表  1  供试引物

    Table  1.   Primes applied

    引物 Primes序列 Sequences(5′-3′)用途 Purpose来源 Sources
    27FAGAGTTTGATCC TGGCTCAG16S rDNA 测序[23]
    1492RACGGTTACCTTGTTACGACTT16S rDNA 测序[23]
    LAD1ACGATGGACTCCAGAG(G/C/A)N(G/C/A)NNNGGAA高效热不对称交错PCR [24]
    LAD3ACGATGGACTCCAGAG(T/A/C)N(A/G/C)NNNCCAC高效热不对称交错PCR [24]
    AC1ACGATGGACTCCAGAG高效热不对称交错PCR[24]
    F389TCAAGCATTTTATCCGTACTCCTG高效热不对称交错PCR[24]
    F536CGGTTGCATTCGATTCCTGTTTGTA高效热不对称交错PCR [24]
    F772TAGGTTGTATTGATGTTGGACGAG高效热不对称交错PCR [24]
    KproFGCAACACTCCGCAATCTATA菌株ACCC 02146启动子序列本研究
    KproRCTCTATCTCCATGAAAGAGT菌株ACCC 02146启动子序列本研究
    下载: 导出CSV

    表  2  菌落形态特征

    Table  2.   Colony morphology

    菌株编号
    Strain No.
    菌落形态特征
    Colony morphology characteristics
    FZSF02圆形凸起,湿润黏稠,红色
    S1圆形凸起,湿润黏稠,红色
    S2圆形凸起,湿润黏稠,红色
    S3圆形凸起,湿润黏稠,红色
    CICC 23703圆形凸起,湿润黏稠,深红色
    CICC 23838圆形凸起,湿润黏稠,红色
    CICC 24478圆形凸起,表面干燥粗糙,红色
    CICC 10698圆形凸起,湿润黏稠,橙红色
    CICC 20223圆形凸起,湿润黏稠,橙色
    CICC 24369圆形凸起,湿润黏稠,深红色
    CCTCC AB2014323圆形凸起,湿润黏稠,深红色
    CCTCC AB2015384圆形凸起,湿润黏稠,红色
    ACCC 02146圆形凸起,湿润黏稠,橙红色
    ACCC 01294圆形凸起,湿润黏稠,深红色
    ACCC 04168圆形凸起,湿润黏稠,深红色
    下载: 导出CSV

    表  3  突变体产灵菌红素能力及其菌量

    Table  3.   Prodigiosin synthesis capabilities and quantity of S.plymuthica mutant

    基因登录号
    Gene accession number
    突变体(基因长度/插入位点)
    Mutant (gene length/insertion site)/bp
    功能
    Function
    OD600OD600
    改变倍数
    OD600 fold change
    OD535OD535改变倍数
    OD535 fold
    change
    OD535改变倍数/
    OD600改变倍数
    OD535 fold change/
    OD600 fold change
    普城沙雷氏菌 ACCC 02146 2.540 1.000 0.213 1.000 1.000
    AEG27540.1 B5-10 (2 673/1 075), B9-2 (2 673/
    1 274), B6-4 (2 673/1 075), B6-6
    (2 673/792), B5-6 (2 673/792),
    B2-11 (2 673/1 266), B5-9 (2 673/153)
    利用磷酸烯醇式丙酮酸酶(PigC 2.880 1.134↑ 0.005 0.023↓ 0.020
    AEG27539.1 B5-1 (2 601/2 116),B8-1 (2 601/2 096), B7-5 (2 601/1 099), B7-6 (2 601/1 099), B7-11 (2 601/1 338), B9-4 (2 601/2 072), B9-5 (2 601/2 071) 假设蛋白(PigD 2.644 1.041↑ 0.005 0.023↓ 0.022
    AEG27535.1 B7-7 (1 971/1 905), B7-1 (1 971/1 905) 甘氨酸C-乙酰转移酶(PigH 2.600 1.025↑ 0.015 0.070↓ 0.068
    AEG27542.1 B5-12 (1 161/195), B5-13 (1 161/794), B6-8 (1 161/35), B7-8 (1 161/35), B9-1 (1 161/35) 异戊酰基-辅酶A脱氢酶(PigA 2.731 1.075↑ 0.004 0.019↓ 0.02
    AEG27541.1 B5-2 (2 031/1 865), B5-11 (2 031/
    1 865), B6-7 (2 031/1 865), B7-12
    (1 161/35)
    富马酸还原酶/琥珀酸脱氢酶黄蛋白结构域蛋白(PigB 2.665 1.049↑ 0.012 0.056↓ 0.053
    AEG30298.1 F5-7 (558/202), F5-4 (558/524), F5-10 (558/273), F5-5 (558/518), F5-6 (558/495), F5-9 (558/189) 麦芽糖O-乙酰转移酶 2.618 1.031↑ 0.037 0.174↓ 0.169
    AEG29267.1 H8-8 (978/910), H8-9 (978/262), H9-8 (978/217) H-醌氧化还原酶亚基1 2.355 0.927↓ 0.245 1.150↑ 1.241
    AEG27501.1 H7-1 (1 011/842), H7-4 (1 011/425), H9-3 (1 011/378), H9-6 (1 011/234) 二氢乳清酸脱氢酶 1.967 0.774↓ 0.589 2.765↑ 3.572
    AEG29268.1 H9-8 (2 748/217), H7-5 (2 748/2 682), H7-9 (2 748/2 041) NADH-醌氧化还原酶G链 2.216 0.872↓ 0.243 1.140↑ 1.307
    AEG26904.1 B9-3 (495/290), B7-2 (495/290), B7-3 (495/290) 肽基脯氨酰异构酶B 3.827 1.507↑ 0.008 0.038↓ 0.025
    AEG26553.1 H8-12 (1 800/1 017), H7-3 (1 800/1 236) 亚硫酸盐还原酶(NADPH)黄蛋白α组分 2.646 1.042↑ 0.269 1.263↑ 1.212
    AEG29408.1 H9-5 (1 728/1 484), H7-11 (1 728/1 484) 磷酸烯醇丙酮酸蛋白磷酸转移酶Ptsl 2.660 1.047↑ 0.574 2.695↑ 2.574
    AEG29263.1 H8-4 (1 848/189), H9-1 (1 848/853) NADH-醌氧化还原酶亚基L 2.473 0.974↓ 0.231 1.085↑ 1.114
    AEG27032.1 H6-5 (390/192) 琥珀酸脱氢酶,细胞色素b556亚基 1.758 0.692↓ 0.230 1.080↑ 1.561
    AEG29294.1 H9-10 (1 518/1 085) 氨基磷酸核糖转移酶 2.317 0.912↓ 0.273 1.282↑ 1.406
    AEG27629.1 H9-9 (1 047/533) 二氢乳清酸酶 2.164 0.852↓ 0.231 1.085↑ 1.273
    AEG27038.1 H8-3 (1 167/336) 琥珀酰辅酶a合成酶(ADP形成)β亚基 2.230 0.878↓ 0.282 1.324↑ 1.508
    AEG27031.1 H8-5 (1 293/402), H8-2 (1 293/0) 柠檬酸合成酶I 2.052 0.808↓ 0.333 1.563↑ 1.934
    AEG29271.1 H8-7 (1 797/618) NADH-醌氧化还原酶亚基C/D 2.542 1.001↑ 0.293 1.376↑ 1.375
    AEG26076.1 H7-7 (1 284/606) 磷酸核糖胺-甘氨酸连接酶 2.579 1.015↑ 0.239 1.122↑ 1.105
    AEG30581.1 F4-1 (1 785/73) ATP结合蛋白 2.640 1.040↑ 0.052 0.244↓ 0.235
    AEG26238.1 B5-3 (630/624) 假设蛋白ALQ63_03 824 1.416 0.557↓ 0.01 0.047↓ 0.084
    AEG29272.1 H7-6 (675/317) H-醌氧化还原酶亚基K 2.342 0.922↓ 0.285 1.338↑ 1.451
    AEG27034.1 H7-8 (1 710/1 665), H9-7 (1 710 /1 352) 琥珀酸脱氢酶或富马酸还原酶黄蛋白亚基 1.603 0.631↓ 0.055 0.258↓ 0.409
    AEG27939.1 B4-1 (435/109), B6-3 (435/109), B6-5 (435/108) MarR家族转录调控因子SlyA 2.712 1.068↑ 0.006 0.028↓ 0.026
    AEG28365.1 F4-4 (849/38), F4-2 (849/36), F4-3 (849/38) winged helix家族双组分转录调控因子RstA 2.71 1.067↑ 0.039 0.183↓ 0.172
    AEG30425.1 H8-1 (633/262) cAMP激活的全局转录调控因子CRP,转录调控因子Crp/Fnr家族 2.243 0.883↓ 0.860 4.038↑ 4.573
    AEF52 157.1 H7-14 (267/3) FAD装配因子SdhE 2.737 1.078↑ 0.259 1.216↑ 1.128
    AEG27490.1 H7-10 (1 110/1 089) 孔蛋白OmpC 1.460 0.575↓ 0.585 2.746↑ 4.776
    AEG26866.1 F5-12 (1 188/864) 多药外排RND转运蛋白质周适配器亚单位SdeX 2.910 1.146↑ 0.022 0.103↓ 0.090
    AEG29409.1 H3-1 (510/364) 葡萄糖转运蛋白亚基IIA_ 2.234 0.880↓ 0.051 0.239↓ 0.272
    下划线标注的为代表性突变体;“↑”代表升高;“↓”代表降低。
    Underline indicates mutant; "↑" indicates elevation; "↓" indicates decline.
    下载: 导出CSV
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  • 收稿日期:  2022-11-10
  • 修回日期:  2023-03-01
  • 网络出版日期:  2023-05-09
  • 刊出日期:  2023-04-28

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