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果生炭疽菌转录因子CfHac1的BRLZ结构域生物学功能研究

李司政 姚权 李河

李司政, 姚权, 李河. 果生炭疽菌转录因子CfHac1的BRLZ结构域生物学功能研究[J]. 北京林业大学学报. doi: 10.12171/j.1000-1522.20210087
引用本文: 李司政, 姚权, 李河. 果生炭疽菌转录因子CfHac1的BRLZ结构域生物学功能研究[J]. 北京林业大学学报. doi: 10.12171/j.1000-1522.20210087
Li Sizheng, Yao Quan, Li He. Functional analysis of BRLZ motif of the transcription factor CfHac1 in Colletotrichum fructicola[J]. Journal of Beijing Forestry University. doi: 10.12171/j.1000-1522.20210087
Citation: Li Sizheng, Yao Quan, Li He. Functional analysis of BRLZ motif of the transcription factor CfHac1 in Colletotrichum fructicola[J]. Journal of Beijing Forestry University. doi: 10.12171/j.1000-1522.20210087

果生炭疽菌转录因子CfHac1的BRLZ结构域生物学功能研究

doi: 10.12171/j.1000-1522.20210087
基金项目: 国家自然科学基金项目(32071765)
详细信息
    作者简介:

    李司政。主要研究方向:森林病理。Email:775394825@qq.com 地址:410004 湖南省长沙市韶山南路498号中南林业科技大学林学院

    责任作者:

    李河,副教授。主要研究方向:森林病理。Email:csuftlihe@163.com 地址:同上

Functional analysis of BRLZ motif of the transcription factor CfHac1 in Colletotrichum fructicola

  • 摘要:   目的  炭疽病是油茶的主要病害,导致巨大的经济损失。果生炭疽菌是油茶炭疽病优势致病菌。对果生炭疽菌的转录因子CfHac1的结构域进行分析,为进一步解析转录因子CfHac1调控病菌致病的分子机制奠定基础,对挖掘油茶炭疽病防控新途径具有重要的理论意义。  方法  采用点突变技术构建转录因子CfHac1结构域缺失载体,通过PEG介导法将该回补载体转化至CfHAC1基因完全缺失突变体的原生质体中,通过博来霉素抗性和荧光筛选获得结构域缺失回补菌株,进一步研究BRLZ结构域在果生炭疽菌中的生物学功能。  结果  结果发现转录因子CfHac1含有一个碱性亮氨酸拉链结构域(BRLZ),该结构域包含58个氨基酸残基;与野生型菌株和完全互补菌株相比,BRLZ结构域缺失突变株ΔCfhac1ΔBRLZ生长速率显著降低,分生孢子产量显著减少,不能形成附着胞,对内质网压力胁迫更敏感,丧失对油茶叶的致病力,其表型与ΔCfhac1突变体一致。  结论  上述结果表明,BRLZ是转录因子CfHac1重要的结构域,对CfHac1在果生炭疽菌中行使正常的生物学功能具有重要的调控作用。

     

  • 图  1  BRLZ结构域缺失突变体获得

    A. CfHac1结构域预测;B.突变体电泳图;M. DL2000 marker;WT. 野生型菌株CFLH16;Δ. ΔCfhac1菌株;ΔBRLZ. ΔCfhac1ΔBRLZ菌株;C. 突变体回补菌株;−. H2O阴性对照. A, domain prediction of CfHac1; B, DNA electrophoretogram of mutants. M, DL2000 marker; WT, wild type strain; Δ, ΔCfhac1 strain; ΔBRLZ, ΔCfhac1ΔBRLZ strain; C, mutant complemented strain; −, H2O negative control.

    Figure  1.  Generation of the BRLZ motif deletion mutants

    图  2  突变体菌株生长速率测定

    A. 各菌株在 PDA、MM培养基上28 ℃黑暗培养3 d的生长情况;CFLH16. 野生型;ΔCfhac1. 突变体;ΔCfhac1ΔBRLZ. 结构域缺失突变体;ΔCfhac1/CfHAC1. 回补菌株;B. 菌落直径差异统计分析;**. 表示差异极显著(P < 0.01)。下同。A. The strains were inoculated on PDA and MM medium at 28 ℃ in the dark for 3 days; CFLH16, wide type; ΔCfhac1, mutant; ΔCfhac1ΔBRLZ, domain deletion mutant; ΔCfhac1/CfHAC1, complemented strains; B, Statistical analysis of the colony diameter; ** represent significant difference (P < 0.01). The same below.

    Figure  2.  Determination of growth rate of mutants

    图  3  菌株突变体对DTT的敏感性测定

    A. 野生型(CFLH16)、突变体(ΔCfhac1)、结构域缺失突变体(ΔCfhac1ΔBRLZ)和回补菌株(ΔCfhac1/CfHAC1)分别接种于PDA培养基以及含有5 mmol/L DTT的PDA培养基上的菌落生长情况;B. 菌株在DTT胁迫培养基中生长抑制率统计分析;误差线采用的是标准偏差. A, The growth of the wild-type strain(CFLH16), CfHAC1 deletion mutant (ΔCfhac1), ΔCfhac1ΔBRLZ and complemented strain (ΔCfhac1/CfHAC1) on PDA plates and PDA plates containing 5 mmol/L DTT; B, Statistical analysis of the growth inhibition rate of strains under DTT stress medium. Error bars represent standard deviations.

    Figure  3.  Sensitivity determination of different strains of mutants to DTT

    图  4  突变体分生孢子数量统计分析

    Figure  4.  The conidial quantitative statistical diagram of mutants

    图  5  突变体致病力测定

    Figure  5.  Pathogenicity test of mutants

    图  6  附着胞形成率统计分析

    Figure  6.  Statistical analysis of the rate of appressorium formation

    表  1  构建BRLZ结构域缺失载体涉及的引物序列

    Table  1.   Primers used of construction of the BRLZ domain deletion vector

    引物名称 Primer name序列(5′—3′) Sequence (5′-3′)用途 Usage
    bZIP13-9F ACTCACTATAGGGCGAATTGGGTACTCA 扩增结构域BRLZ缺失回补序列
    Amplify ΔBRLZ complemented sequence
    AATTGGTTCGCGTAGCACTGTAGCAGAA
    bZIP13-10R CACCACCCCGGTGAACAGCTCCTCGCCC 扩增结构域BRLZ缺失回补序列
    Amplify ΔBRLZ complemented sequence
    TTGCTCACAGCTAATCGACAACGCTTCC
    bZIP13-BRR1 TTCCGTCTTCGCTCTTTTCC 扩增结构域BRLZ缺失回补序列
    Amplify ΔBRLZ complemented sequence
    bZIP13-BRF2 GGAAAAGAGCGAAGACGGAAAAATTTC 扩增结构域BRLZ缺失回补序列
    Amplify ΔBRLZ complemented sequence
    GCCGTGACTCTGG
    GFP-R GACACGCTGAACTTGTGGCCGTT 验证回补载体序列 Validation of complemented sequence
    bZIP13-atgF ATGGCTGCTTGGGAACAGAC 验证结构域缺失序列 Validation of ΔBRLZ domain sequence
    bZIP13-tgaR AGCTAATCGACAACGCTTCC 验证结构域缺失序列 Validation of ΔBRLZ domain sequence
    下载: 导出CSV
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  • 收稿日期:  2021-03-11
  • 修回日期:  2021-05-05
  • 网络出版日期:  2021-06-21

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