尖孢镰刀菌黏团专化型转录因子SNT2基因敲除及其功能分析

doi:10.7668/hbnxb.20195567

摘要/Abstract

摘要：

甘蓝枯萎病是由尖孢镰刀菌黏团专化型(FOC)引起的土传真菌病害,严重影响甘蓝产量和品质。为了明确该病原菌中转录因子SNT2的生物学功能,利用同源重组和原生质体转化技术,成功获得了尖孢镰刀菌中SNT2基因敲除突变体ΔSNT2,并对其表型和致病力进行了分析。结果表明:尖孢镰刀菌中SNT2编码1 529个氨基酸,具有与DNA结合的SANT结构域,蛋白归属亲水性蛋白质。与野生型菌株相比,ΔSNT2突变体菌丝生长速率降低且分隔明显增加,分生孢子产孢量显著下降。基于外源胁迫结果显示,ΔSNT2在1 mol/L 山梨醇的渗透压胁迫中表现不敏感,但对0.1% H₂O₂、2 mol/L NaCl和0.05% 刚果红(CR)的氧胁迫、盐胁迫和细胞壁胁迫的耐受性降低,同时致病力测定结果表明,接种ΔSNT2突变体的病情指数显著低于接种野生型,SNT2的缺失导致尖孢镰刀菌致病力显著下降。综上,转录因子SNT2在病原菌与寄主互作过程中对于维持细胞壁的完整性具有重要作用,同时参与调控尖孢镰刀菌的生长发育和致病力。

关键词: 尖孢镰刀菌, SNT2, 表型, 致病力, 功能分析, 基因敲除

Abstract:

Cabbage fusarium wilt is a serious soil-borne fungal disease caused by Fusarium oxysporum f.sp.conglutinans (FOC),which affects the yield and quality of cabbage.In order to clarify the biological function of the transcription factor SNT2 in this pathogen,the SNT2 gene knockout mutant ΔSNT2 in F.oxysporum was successfully obtained by homologous recombination and protoplast transformation,and its phenotype and pathogenicity were analyzed.The results showed that SNT2 in F.oxysporum encoded 1 529 amino acids,had a SANT domain that binds to DNA,and the protein belonged to a hydrophilic protein.Compared with the wild type strain,the mycelial growth rate of the ΔSNT2 mutant decreased and the septation increased significantly,and the conidial production decreased significantly.Based on the results of exogenous stress,ΔSNT2 was insensitive to osmotic stress of 1 mol/L sorbitol,but its tolerance to oxygen stress,salt stress and cell wall stress of 0.1% H₂O₂,2 mol/L NaCl and 0.05% Congo red was reduced.At the same time,the pathogenicity test showed that the disease index of ΔSNT2 mutant was significantly lower than that of wild type,and the deletion of SNT2 resulted in a significant decrease in the pathogenicity of F.oxysporum.In conclusion,the transcription factor SNT2 plays an important role in maintaining the integrity of the cell wall during the interaction between the pathogen and the host,and participates in the regulation of the growth and development of F.oxysporum and the expression of pathogenicity.

Key words: Fusarium oxysporum, SNT2, Phenotype, Pathogenicity, Functional analysis, Gene knockout

徐新瑞, 宋泽龙, 李二峰. 尖孢镰刀菌黏团专化型转录因子SNT2基因敲除及其功能分析[J]. 华北农学报, 2025, 40(2): 173-180. doi: 10.7668/hbnxb.20195567.

XU Xinrui, SONG Zelong, LI Erfeng. Knockout and Functional Analysis of Transcription Factor SNT2 Coding Gene in Fusarium oxysporum f.sp.conglutinans[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(2): 173-180. doi: 10.7668/hbnxb.20195567.

http://www.hbnxb.net/CN/Y2025/V40/I2/173

图/表 8

表1 引物名称及序列

Tab.1 Primer names and sequences

引物名称 Primer name	引物序列(5'—3') Primer sequences(5'—3')
up-F	AAATGCGGATGACTTGGCG
up-R	ACCTCCACTAGCTCCAGCCAAGAGTT GCGAAGGATGAAGACGG
down-F	GAATAGAGTAGATGCCGACCGGGTG AAAGTCTAAGAGGGCGAGC
down-R	AGTGCGAGAGAGCGGTATTT
hygR-F1	CTTGGCTGGAGCTAGTGGAGGT
hygR-R1	GGATGCCTCCGCTCGAAGTA
hygR-F2	CGTTGCAAGACCTGCCTGAA
hygR-R2	CCCGGTCGGCATCTACTCTATTC
upcheck-F	ATTGCTGTGTTGGTGCGTC
upcheck-R	ATTGCTGTGTTGGTGCGTC
downcheck-F	TCTGGACCGATGGCTGTGTAG
downcheck-R	CGTGTGTTGTTTCTCTCACCAT

图1 同源重组敲除策略

Fig.1 Homologous recombination knockout strategy

图2 SNT2蛋白结构域预测

Fig.2 SNT2 protein domain prediction

图3 同源置换臂的扩增 M.DNA Marker;A.up片段;B.down片段;C.hygR1片段; D.hygR2片段;E.up-hygR1片段;F.hygR2-down片段。

Fig.3 Amplification of homologous replacement arms M.DNA Marker;A.up fragment;B.down fragment;C.hygR1 fragment; D.hygR2 fragment;E.up-hygR1 fragment;F.hygR2-down fragment.

图4 缺失突变体的PCR和Southern杂交鉴定 A.PCR鉴定:M1.DNA Marker。B.敲除突变体鉴定:M2.DNA Marker。

Fig.4 PCR and Southern Blot analysis of the deletion mutant A.PCR identification:M1.DNA Marker.B.Knockout mutant identification:M2.DNA Marker.

图5 野生型和突变体的菌丝生长和产孢量比较 A.野生型和突变体的菌落生长状况;B.野生型和突变体的菌丝隔膜长度;C.野生型和突变体的菌丝形态(m₁、m₂、m₃和n₁、n₂、n₃表示菌丝隔膜长度);D.野生型和突变体的产孢量。不同小写字母代表差异显著(P<0.05)。图6,7同。

Fig.5 Comparison of mycelial growth and asexual reproduction between wild type and mutants A.Colony growth analysis between wild-type and mutants;B.The hyphal septum length of wild type and mutant;C.Mycelial morphology analysis between wild-type and mutants(m₁,m₂,m₃ and n₁,n₂,n₃ represent the length of hyphal septum);D.Sporulation between wild-type and mutants.Different lowercase letters represent significant differences(P<0.05).The same as Fig.6,7.

图6 野生型和突变体的生长胁迫比较 A.不同胁迫条件下野生型和突变体的菌落生长情况;B.相对抑制率。

Fig.6 Comparison of growth stress between wild type and mutants A.Colonies growth analysis under different stresses;B.Relative inhibitory rate.

图7 野生型和突变体的致病力比较 A.接种野生型和突变体后6~15 d内甘蓝的病情指数;B.接种野生型和突变体11 d后甘蓝的生长状况:上排为叶片病害症状特写图,下排为植株病害症状图。

Fig.7 Comparison of pathogenicity between wild type and mutant A.The disease index of wild-type and mutant on cabbage from 6 to 15 days after inoculation;B.The growth status of cabbage inoculated with wild type and mutant on Day 11:the upper row is the leaf disease symptom feature map,and the lower row is the plant disease symptom map.

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