香蕉枯萎病菌4号生理小种β2-微管蛋白基因敲除与表型分析
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摘要
【目的】克隆并鉴定香蕉枯萎病菌(Foc4)β2-微管蛋白(β2-tub)基因,阐明β2-tub在多菌灵的抗药性及在病原菌致病过程中发挥的作用。【方法】采用PCR技术克隆了β2-tub的序列全长,并对其进行生物信息学分析。应用Splitmarker同源重组技术获得Foc4的β2-tub基因敲除突变体,并对其突变体的生物学表型、致病力及其对多菌灵的敏感性进行测定。【结果】生物信息学分析表明,Foc4β2-tub基因全长为1 694 bp,cDNA编码区全长1 347 bp,由4个内含子和5个外显子组成,编码蛋白含448个氨基酸。Foc4对多菌灵表现为敏感(EC50=0.51μg·mL~(-1)),与Foc4相比,β2-tub基因敲除突变体对多菌灵的敏感性(EC50=0.36μg·mL~(-1))表现显著增强,差异显著(p <0.05)。与Foc4相比,β2-tub基因敲除突变体的生物学表型没有变化,对巴西蕉苗的致病力明显减弱。【结论】β2-tub基因具有高度保守性,β2-tub基因敲除突变体对细胞壁选择性压力、氧化压力和渗透压力均没有影响,但致病力下降,同时β2-tub基因的变化会引起Foc4对多菌灵抗性的改变。
【Objective】The objective of this study was to clone and identify the β2-tubulin(β2-tub)gene of Fusarium oxysporum f. sp. cubense race 4(Foc4), and reveal the function of β2-tub in resistance of Foc4 to carbendazim and its role in the pathogenic process of Foc4.【Methods】Based on the sequences of β2-tub gene(XM_011327927.1) of Fusarium graminearum and the complete genome sequence of the Foc4 54006 strain(JH658279.1), specific primers were designed. A full-length sequence of β2-tub gene from Foc4 was cloned by PCR amplification, and sequence characterization, phylogenetic clustering and protein domains of β2-tub gene were also analyzed, respectively. The signature domains were analyzed by SMART software and the phylogenetic tree was built by MEGA 6.0. β2-tub knockout mutants of Foc4 were obtained by split-marker homologous recombination technology. Sensitivity of Foc4 and its knockout mutants to carbendazim was tested by inoculating their mycelium onto PDA plates with different concentrations of carbendazim, respectively. The biological phenotypes in-cluding the growth rate of colonies, the amount of sporulation, the form of mycelium, selective pressure of cell wall, oxidation pressure and osmotic pressure of Foc4 and its knockout mutants were tested. In addition, the pathogenicity of Foc4 and its knockout mutants was tested by pot culture root drenching method. Foc4 and its knockout mutants were treated with 15 Brazil banana seedlings(Cavendish AAA).Each seedling was irrigated with 20 mL spores suspension, and aseptic water was used as a control. The corm of Brazil banana was cut longitudinally after 30 d, and the browning degree of the bulb and the condition of the chlorosis of the outer leaves were observed.【Results】Bioinformatics analysis revealed that the complete DNA and cDNA sequences of β2-tub from Foc4 were 1 694 bp and 1 347 bp, respectively. The cDNA of β2-tub included 5 exons and 4 introns and encoded a protein of 448 amino-acids.Moreover, the β2-tub was highly conserved. Compared with other plant pathogenic fungi, the gene was highly homologous. In Foc4, the β1-tub protein encoded 446 amino-acids. The homology of DNA sequence of β1-tub and β2-tub was 64.67%, the homology of amino acid sequence was 76.34%, and they had the same domain. The phylogenetic tree showed that β2-tub was closely related to F. oxysporum f.sp. lycopersici and F. graminearum. The β2-tub gene is highly conserved in biological evolution with other common plant pathogenic fungi. After two rounds of PCR amplification, the upstream and downstream recombination fragments required for homologous recombination for protoplast transformation were successfully amplified by split-marker. The sensitivity test of Foc4 and its knockout mutants to carbendazim showed that the β2-tub gene was related to the resistance of Carbendazim. The EC50 value of Foc4 to carbendazim was 0.51 μg · mL~(-1), and the EC50 values of β2-tub gene knockout mutant to carbendazim was 0.36 μg · mL~(-1). Both were sensitive to carbendazim, however, the sensitivity to carbendazim of β2-tub gene knockout mutant increased significantly. Compared with Foc4, the growth rate, the sporulation quantity and the mycelial morphology of the β2-tub gene knockout mutant were not changed,and the difference was not significant. Sensitivity analysis of stress factors showed that β2-tub gene knockout mutants had no influence on cell wall selective pressure, oxidative stress and osmotic pressure. The results of pathogenicity test showed that the leaves of the plants inoculated with wild strains were yellow and wilted, most of which were yellow in the lower leaves, and the browning of the bulbs was serious by the incision of the bulbs. However, a few leaves of the plants inoculated with the β2-tub gene knockout mutant strain were yellow, and the bulbs appeared slightly browning. There was no leaf yellowing in the control plants inoculated with H2 O, and no browning in the corms. According to the disease index statistics, the results showed that the average disease index(42.24) of the inoculated strain ofβ2-tub gene knockout mutant was significantly lower than the average disease index(59.17) of Foc4 wild fungi.【Conclusion】Foc4 wild strains and β2-tub gene knockout mutant strains all maintained high sensitivity to carbendazim. Furthermore, the knockout mutants showed a significant increase in sensitivity to carbendazim, indicating that the change of β2-tub gene caused the resistance of wild strain to carbendazim. The gene of β2-tub was highly conserved and had a typical microtubule protein domain. The biological phenotypes of β2-tub knockout mutants, including growth rate, sporulation, hyphae morphology, selective pressure on cell wall, oxidative pressure and osmotic pressure, were not significantly different, but their pathogenicity to Brazil banana seedlings decreased significantly.
【Objective】The objective of this study was to clone and identify the β2-tubulin(β2-tub)gene of Fusarium oxysporum f. sp. cubense race 4(Foc4), and reveal the function of β2-tub in resistance of Foc4 to carbendazim and its role in the pathogenic process of Foc4.【Methods】Based on the sequences of β2-tub gene(XM_011327927.1) of Fusarium graminearum and the complete genome sequence of the Foc4 54006 strain(JH658279.1), specific primers were designed. A full-length sequence of β2-tub gene from Foc4 was cloned by PCR amplification, and sequence characterization, phylogenetic clustering and protein domains of β2-tub gene were also analyzed, respectively. The signature domains were analyzed by SMART software and the phylogenetic tree was built by MEGA 6.0. β2-tub knockout mutants of Foc4 were obtained by split-marker homologous recombination technology. Sensitivity of Foc4 and its knockout mutants to carbendazim was tested by inoculating their mycelium onto PDA plates with different concentrations of carbendazim, respectively. The biological phenotypes in-cluding the growth rate of colonies, the amount of sporulation, the form of mycelium, selective pressure of cell wall, oxidation pressure and osmotic pressure of Foc4 and its knockout mutants were tested. In addition, the pathogenicity of Foc4 and its knockout mutants was tested by pot culture root drenching method. Foc4 and its knockout mutants were treated with 15 Brazil banana seedlings(Cavendish AAA).Each seedling was irrigated with 20 mL spores suspension, and aseptic water was used as a control. The corm of Brazil banana was cut longitudinally after 30 d, and the browning degree of the bulb and the condition of the chlorosis of the outer leaves were observed.【Results】Bioinformatics analysis revealed that the complete DNA and cDNA sequences of β2-tub from Foc4 were 1 694 bp and 1 347 bp, respectively. The cDNA of β2-tub included 5 exons and 4 introns and encoded a protein of 448 amino-acids.Moreover, the β2-tub was highly conserved. Compared with other plant pathogenic fungi, the gene was highly homologous. In Foc4, the β1-tub protein encoded 446 amino-acids. The homology of DNA sequence of β1-tub and β2-tub was 64.67%, the homology of amino acid sequence was 76.34%, and they had the same domain. The phylogenetic tree showed that β2-tub was closely related to F. oxysporum f.sp. lycopersici and F. graminearum. The β2-tub gene is highly conserved in biological evolution with other common plant pathogenic fungi. After two rounds of PCR amplification, the upstream and downstream recombination fragments required for homologous recombination for protoplast transformation were successfully amplified by split-marker. The sensitivity test of Foc4 and its knockout mutants to carbendazim showed that the β2-tub gene was related to the resistance of Carbendazim. The EC50 value of Foc4 to carbendazim was 0.51 μg · mL~(-1), and the EC50 values of β2-tub gene knockout mutant to carbendazim was 0.36 μg · mL~(-1). Both were sensitive to carbendazim, however, the sensitivity to carbendazim of β2-tub gene knockout mutant increased significantly. Compared with Foc4, the growth rate, the sporulation quantity and the mycelial morphology of the β2-tub gene knockout mutant were not changed,and the difference was not significant. Sensitivity analysis of stress factors showed that β2-tub gene knockout mutants had no influence on cell wall selective pressure, oxidative stress and osmotic pressure. The results of pathogenicity test showed that the leaves of the plants inoculated with wild strains were yellow and wilted, most of which were yellow in the lower leaves, and the browning of the bulbs was serious by the incision of the bulbs. However, a few leaves of the plants inoculated with the β2-tub gene knockout mutant strain were yellow, and the bulbs appeared slightly browning. There was no leaf yellowing in the control plants inoculated with H2 O, and no browning in the corms. According to the disease index statistics, the results showed that the average disease index(42.24) of the inoculated strain ofβ2-tub gene knockout mutant was significantly lower than the average disease index(59.17) of Foc4 wild fungi.【Conclusion】Foc4 wild strains and β2-tub gene knockout mutant strains all maintained high sensitivity to carbendazim. Furthermore, the knockout mutants showed a significant increase in sensitivity to carbendazim, indicating that the change of β2-tub gene caused the resistance of wild strain to carbendazim. The gene of β2-tub was highly conserved and had a typical microtubule protein domain. The biological phenotypes of β2-tub knockout mutants, including growth rate, sporulation, hyphae morphology, selective pressure on cell wall, oxidative pressure and osmotic pressure, were not significantly different, but their pathogenicity to Brazil banana seedlings decreased significantly.
引文
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[2] HWANG S C,KOW H. Cavendish banana cultivars resistant to fusarium wilt acquired through somaclonal variation in Taiwan[J]. Plant Disease,2004,88(6):580-588.
[3]彭埃天,宋晓兵,凌金锋,刘景梅.香蕉枯萎病菌4号生理小种分子检测与枯萎病生物防治研究进展[J].果树学报,2009,26(1):77-81.PENG Aitian,SONG Xiaobing,LING Jinfeng,LIU Jingmei. Advances in research on molecular detection and biological control of Fusarium oxysporum f. sp. cubense race 4[J]. Journal of Fruit Science,2009,26(1):77-81.
[4]郭立佳,杨腊英,彭军,王国芬,梁昌聪,刘磊,黄俊生.不同药剂防治香蕉枯萎病效果评价[J].中国农学通报,2013,29(1):188-192.GUO Lijia,YANG Laying,PENG Jun,WANG Guofen,LIANG Changcong,LIU Lei,HUANG Junsheng. Evaluation of control affection of fungicides on Fusarium Wilt of banana[J]. Chinese Agricultural Science Bulletin,2013,29(1):188-192.
[5]宁家相,王振通.几种药剂对香蕉枯萎病防治效果研究[J].农家科技旬刊,2016(1):1.NING Jiaxiang,WANG Zhentong. Study on the effect of prevention and control of several chemicals on Fusarium Wilt of banana[J]. Farm Technology Magazine,2016(1):1.
[6] LIN B,SHEN H. Fusarium oxysporum,f. sp. cubense[M]. Singapore:Springer,2017.
[7]齐兴柱,杨腊英,黄俊生. FOC4的2个过氧化氢酶基因的克隆与表达分析及其引起的香蕉苗活性氧迸发研究[J].中国农学通报,2012,28(15):163-169.QI Xingzhu,YANG Laying,HUANG Junsheng. Cloning and expression analysis of two catalase gene of FOC4 and the study on the oxidative burst in banana by FOC4[J]. Chinese Agricultural Science Bulletin,2012,28(15):163-169.
[8]王小琳,李春强,杨景豪,李文彬,孙建波,彭明.香蕉枯萎病菌4号生理小种cat1基因敲除与表型分析[J].热带作物学报,2017,38(2):335-342.WANG Xiaolin,LI Chunqiang,YANG Jinghao,LI Wenbin,SUN Jianbo,PENG Ming. Gene knockout and phenotype analysis of the catalase-1 in Fusarium oxysporum f. sp. cubense Race4[J]. Chinese Journal of Tropical Crops,2017,38(2):335-342.
[9] DING Z J,LI M,SUN F,XI P G,SUN L H,ZHANG L H,JIANG Z D. Mitogen-activated protein kinases are associated with the regulation of physiological traits and virulence in Fusarium oxysporum f. sp. cubense[J]. PLoS One,2015,10(4):e0122634.
[10] GUO L J,YANG L Y,LIANG C C,WANG J,LIU L,HUANG J S. The G-protein subunits FGA2 and FGB1 play distinct roles in development and pathogenicity in the banana fungal pathogen Fusarium oxysporum f. sp. cubense[J]. Physiological&Molecular Plant Pathology,2016,93:29-38.
[11] QI X,GUO L J,YANG L Y,HUANG J S. Foatf1,a bZIP transcription factor of Fusarium oxysporum f. sp. cubense,is involved in pathogenesis by regulating the oxidative stress responses of Cavendish banana(Musa spp.)[J]. Physiological&Molecular Plant Pathology,2013,84(5):76-85.
[12]林兰稳,奚伟鹏,黄赛花.香蕉镰刀菌枯萎病防治药剂的筛选[J].生态环境学报,2003,12(2):182-183.LIN Lanwen,XI Weipeng,HUANG Saihua. Screening of preventive fungicide of Fusarium oxysporum f. sp. cubense[J].Ecology and Environment,2003,12(2):182-183.
[13]王芳,李静,洪文兴.几种杀菌剂对香蕉枯萎病菌的抑制作用[J].湖北农业科学,2009,48(1):100-101.WANG Fang,LI Jing,HONG Wenxing. Antifungal activity of several fungicides to Fusarium oxysporium f. sp. cubense from banana[J]. Hubei Agricultural Science,2009,48(1):100-101.
[14] QIU J B,HUANG T T,XU J Q,BI C W,CHEN C J,ZHOU M G.β-Tubulins in Gibberella zeae:their characterization and contribution to carbendazim resistance[J]. Pest Management Science,2012,68(8):1191-1198.
[15] CHEN C J,YU J J,BI C W,ZHANG Y N,XU J Q,WANG J X.Mutations in a beta-tubulin confer resistance of Gibberella zeae to benzimidazole fungicides[J]. Phytopathology,2009,99(12):1403-1411.
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