Functional analysis of BRLZ motif of the transcription factor CfHac1 in Colletotrichum fructicola
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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。 Abstract:Objective Colletotrichum fructicola is a major pathogen causing anthracnose on Camellia oleifera, and leads to substantial losses annually. The domain analysis of transcription factor CfHac1 will help us understanding the CfHac1-regulated pathogenic mechanism of the pathogen and providing new insights to control this disease. Method We constructed the domain deletion vector by using point mutation technology and introduced it into the ΔCfhac1 mutant through the PEG-mediated protoplast transformation. The strains were selected by bleomycin and fluorescence, then functional analyzed in C. fructicola. Result The structure prediction revealed that CfHac1 contains one basic region-leucine zipper motif (BRLZ), which contains 58 amino acid residues. Comparing to the wide type and complemented strains, the mycelial growth rate, conidiation and appressorium formation rate of the Cfhac1ΔBRLZ were significantly reduced, and the Cfhac1ΔBRLZ strain was sensitive to dithiothreitol. The pathogenicity test showed that the Cfhac1ΔBRLZ lost the ability to infect C. oleifera leaves, which phenotype is consistent with ΔCfhac1 mutant. Conclusion The results showed that BRLZ is an important domain of CfHac1 and is essential for normal function of CfHac1 in C. fructicola. -
Key words:
- Camellia oleifera /
- anthracnose /
- Colletotrichum fructicola /
- CfHac1 /
- BRLZ motif /
- pathogenicity
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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
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 sequenceAATTGGTTCGCGTAGCACTGTAGCAGAA bZIP13-10R CACCACCCCGGTGAACAGCTCCTCGCCC 扩增结构域BRLZ缺失回补序列
Amplify ΔBRLZ complemented sequenceTTGCTCACAGCTAATCGACAACGCTTCC bZIP13-BRR1 TTCCGTCTTCGCTCTTTTCC 扩增结构域BRLZ缺失回补序列
Amplify ΔBRLZ complemented sequencebZIP13-BRF2 GGAAAAGAGCGAAGACGGAAAAATTTC 扩增结构域BRLZ缺失回补序列
Amplify ΔBRLZ complemented sequenceGCCGTGACTCTGG 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 
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