哈茨木霉C2H2型转录因子Tha09974功能研究
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摘要
哈茨木霉Th33菌株能拮抗多种植物病原真菌,具有重要的研究和应用价值。实验室前期研究了哈茨木霉Th33 Gα蛋白基因Thga1的功能,发现Thga1敲除突变株的分生孢子梗和分生孢子量显著减少。对该敲除株和Th33进行转录组测序,发现共有29个转录因子发生显著差异表达。本文选取差异表达量最大的C2H2型转录因子基因Tha09974进行功能研究。构建了Tha09974敲除突变株Δ9974及其回复突变株R9974,对野生菌Th33、Δ9974和R9974分别进行了生长、产孢及拮抗特性检测。结果显示,与野生菌Th33相比,Δ9974的菌落生长速度没有显著差异,菌丝生物量降低了51.5%,产孢量降低了73.6%;Δ9974较野生菌Th33对番茄灰霉病菌Botrytis cinerea Pers.和黄瓜枯萎病菌Fusarium oxysporum f. sp. cucumerinum的生长抑制率分别降低了41.67%和45.15%。R9974和Th33的生长速度、生物量、产孢量及对番茄灰霉病菌的抑制率没有显著差异,对黄瓜枯萎病菌的抑制率低于野生菌Th33,但高于Δ9974。以上结果说明,Tha09974能够正调控Th33的生长、产孢和对病原菌的拮抗能力,并受到Thga1的正调控。本研究为进一步揭示木霉菌的产孢调控机制奠定了基础。
Trichoderma harzianum Th33 can effectively antagonize many plant pathogenic fungi and has important research and application value. In our previous study, a signal transfer protein Gα gene Thga1 was knocked out from Th33, and the amount of conidiophores and conidia of the knock-out mutant was significantly decreased.Transcriptome sequencing of the Thga1 knockout mutant and Th33 revealed that a total of 29 transcription factors(TFs) were significantly differentially expressed. Among those TFs, a C2 H2 transcription factor Tha09974 gene showed the largest differential expression ratio. In this study, Tha09974 was cloned, and Tha09974 knock-out mutant Δ9974 and its reverse mutant R9974 were constructed. The growth and sporulation characters and antagonism against Botrytis cinerea and Fusarium oxysporum of the wild Th33, Δ9974 and R9974 were analyzed.Compared to the wild Th33, the mycelial growth rate of Δ9974 was not significantly different with that of wild type Th33, while the biomass and spore production of Δ9974 decreased by 51.5% and 73.6%, respectively, and the growth inhibition rates against B. cinerea and F. oxysporum decreased by 41.67% and 45.15%, respectively. There was no significant difference in growth rate, biomass, sporulation and the inhibition rate on B. cinerea with R9974 and Th33, but the inhibition rate of R9974 against F. oxysporum was lower than that of the wild strain Th33, while higher than Δ9974. The above results indicated that the expression of Tha09974 was positively regulated by Thga1,and can regulate the growth, sporulation and antagonism of Th33. This study laid the foundation for further revealing the sporulation regulation mechanism of Trichoderma.
Trichoderma harzianum Th33 can effectively antagonize many plant pathogenic fungi and has important research and application value. In our previous study, a signal transfer protein Gα gene Thga1 was knocked out from Th33, and the amount of conidiophores and conidia of the knock-out mutant was significantly decreased.Transcriptome sequencing of the Thga1 knockout mutant and Th33 revealed that a total of 29 transcription factors(TFs) were significantly differentially expressed. Among those TFs, a C2 H2 transcription factor Tha09974 gene showed the largest differential expression ratio. In this study, Tha09974 was cloned, and Tha09974 knock-out mutant Δ9974 and its reverse mutant R9974 were constructed. The growth and sporulation characters and antagonism against Botrytis cinerea and Fusarium oxysporum of the wild Th33, Δ9974 and R9974 were analyzed.Compared to the wild Th33, the mycelial growth rate of Δ9974 was not significantly different with that of wild type Th33, while the biomass and spore production of Δ9974 decreased by 51.5% and 73.6%, respectively, and the growth inhibition rates against B. cinerea and F. oxysporum decreased by 41.67% and 45.15%, respectively. There was no significant difference in growth rate, biomass, sporulation and the inhibition rate on B. cinerea with R9974 and Th33, but the inhibition rate of R9974 against F. oxysporum was lower than that of the wild strain Th33, while higher than Δ9974. The above results indicated that the expression of Tha09974 was positively regulated by Thga1,and can regulate the growth, sporulation and antagonism of Th33. This study laid the foundation for further revealing the sporulation regulation mechanism of Trichoderma.
引文
[1]Afa S,Sajad M,Nazaruddin N,et al.MicroRNA and transcription factor:key players in plant regulatory network[J].Frontiers in Plant Science,2017,8:e565.
[2]Kim H R,Chae K S,Han K H,et al.The nsdc gene encoding a putative C2H2-type transcription factor is a key activator of sexual development in Aspergillus nidulans[J].Genetics,2009,182(3):771-783.
[3]李慧燕,林良才,肖冬光,等.粗糙脉孢菌C2H2转录因子家族基因敲除突变体产纤维素酶筛选分析[J].菌物学报,2016,35(2):161-169.
[4]Weindling R.Trichoderma lignorum as a parasite of other soil fungi[J].Phytopathology,1932,22(8):837-845.
[5]Manczinger L,Antal Z,Kredics L.Ecophysiology and breeding of mycoparasitic trichoderma strains(a review)[J].Acta Microbiologica et Immunologica Hungarica,2002,49(1):1-14.
[6]Li L,Wright S J,Krystofova S,et al.Heterotrimeric g protein signaling in filamentous fungi[J].Annual Review of Microbiology,2007,61:423-452.
[7]Qing S,Xiliang J,Li P,et al.Functions of thga1 gene in Trichoderma harzianum based on transcriptome analysis[J].BioMed Research International,2016,e8329513.
[8]鲍龙飞,秦玉琪,曲音波.Flug-brla途径参与构巢曲霉无性发育机制的研究进展[J].微生物学通报,2014,41(1):104-110.
[9]Hu P,Wang Y,Zhou J,et al.Acstua,which encodes an apses transcription regulator,is involved in conidiation,cephalosporin biosynthesis and cell wall integrity of Acremonium chrysogenum[J].Fungal Genetics and Biology,2015,83:26-40.
[10]Yamada O,Lee B R,Gomi K,et al.Cloning and functional analysis of the aspergillus oryzae conidiation regulator gene brla by its disruption and misscheduled expression[J].Journal of Bioscience and Bioengineering,1999,87(4):424-429.
[11]孙青,蒋细良,庞丽,等.哈茨木霉Th33全基因组序列测定与分析[J].中国生物防治学报,2016,32(2):205-214.
[12]冀颖,李梅,田云龙,等.哈茨木霉几丁质合酶基因thchsc的克隆及序列分析[J].中国农业科学,2011,44(17):3537-3546.
[13]牛静,李梅,范亮波,等.哈茨木霉caf类似基因thcon1的克隆及功能初探[J].中国生物防治学报,2013,29(1):89-96.
[14]黄亚丽,蒋细良,田云龙,等.根癌农杆菌介导的哈茨木霉菌遗传转化的研究[J].中国生物工程杂志,2008,28(3):38-43.
[15]刘增亮,李梅,张玉杰,等.哈茨木霉thga1基因敲除突变株对几种植物病原真菌的离体拮抗作用[J].中国生物防治学报,2014,30(3):370-375.
[16]刘少帅.稻瘟菌C3HC和C2H2型锌指蛋白(ZFC3、ZFC2)参与MoLon1致病过程的机理研究[D].长春:吉林大学,2016.
[17]Tian L,Yu J,Wang Y,et al.The C2H2 transcription factor vdmsn2 controls hyphal growth,microsclerotia formation,and virulence of Verticillium dahliae[J].Fungal Biology,2017,121(12):1001-1010.
[18]石佑慧.蝗绿僵菌MabrlA在产孢调控作用和致病机制中的功能研究[D].重庆:重庆大学,2017.
[19]Carreras Villase?or H,Sánchez Arreguín J A,Herreraestrella A H.Trichoderma:sensing the environment for survival and dispersal[J].Microbiology,2012,158(1):3-16.
[20]Jie M,Wang L,Jiang X,et al.Functions of the C2H2 transcription factor gene thmea1 in Trichoderma harzianum under copper stress based on transcriptome analysis[J].BioMed Research International,2018,e8149682.
[21]Kuo J S,Patel M,Gamse J,et al.Opl:a zinc finger protein that regulates neural determination and patterning in xenopus[J].Development,1998,125(15):2867-2882.
[2]Kim H R,Chae K S,Han K H,et al.The nsdc gene encoding a putative C2H2-type transcription factor is a key activator of sexual development in Aspergillus nidulans[J].Genetics,2009,182(3):771-783.
[3]李慧燕,林良才,肖冬光,等.粗糙脉孢菌C2H2转录因子家族基因敲除突变体产纤维素酶筛选分析[J].菌物学报,2016,35(2):161-169.
[4]Weindling R.Trichoderma lignorum as a parasite of other soil fungi[J].Phytopathology,1932,22(8):837-845.
[5]Manczinger L,Antal Z,Kredics L.Ecophysiology and breeding of mycoparasitic trichoderma strains(a review)[J].Acta Microbiologica et Immunologica Hungarica,2002,49(1):1-14.
[6]Li L,Wright S J,Krystofova S,et al.Heterotrimeric g protein signaling in filamentous fungi[J].Annual Review of Microbiology,2007,61:423-452.
[7]Qing S,Xiliang J,Li P,et al.Functions of thga1 gene in Trichoderma harzianum based on transcriptome analysis[J].BioMed Research International,2016,e8329513.
[8]鲍龙飞,秦玉琪,曲音波.Flug-brla途径参与构巢曲霉无性发育机制的研究进展[J].微生物学通报,2014,41(1):104-110.
[9]Hu P,Wang Y,Zhou J,et al.Acstua,which encodes an apses transcription regulator,is involved in conidiation,cephalosporin biosynthesis and cell wall integrity of Acremonium chrysogenum[J].Fungal Genetics and Biology,2015,83:26-40.
[10]Yamada O,Lee B R,Gomi K,et al.Cloning and functional analysis of the aspergillus oryzae conidiation regulator gene brla by its disruption and misscheduled expression[J].Journal of Bioscience and Bioengineering,1999,87(4):424-429.
[11]孙青,蒋细良,庞丽,等.哈茨木霉Th33全基因组序列测定与分析[J].中国生物防治学报,2016,32(2):205-214.
[12]冀颖,李梅,田云龙,等.哈茨木霉几丁质合酶基因thchsc的克隆及序列分析[J].中国农业科学,2011,44(17):3537-3546.
[13]牛静,李梅,范亮波,等.哈茨木霉caf类似基因thcon1的克隆及功能初探[J].中国生物防治学报,2013,29(1):89-96.
[14]黄亚丽,蒋细良,田云龙,等.根癌农杆菌介导的哈茨木霉菌遗传转化的研究[J].中国生物工程杂志,2008,28(3):38-43.
[15]刘增亮,李梅,张玉杰,等.哈茨木霉thga1基因敲除突变株对几种植物病原真菌的离体拮抗作用[J].中国生物防治学报,2014,30(3):370-375.
[16]刘少帅.稻瘟菌C3HC和C2H2型锌指蛋白(ZFC3、ZFC2)参与MoLon1致病过程的机理研究[D].长春:吉林大学,2016.
[17]Tian L,Yu J,Wang Y,et al.The C2H2 transcription factor vdmsn2 controls hyphal growth,microsclerotia formation,and virulence of Verticillium dahliae[J].Fungal Biology,2017,121(12):1001-1010.
[18]石佑慧.蝗绿僵菌MabrlA在产孢调控作用和致病机制中的功能研究[D].重庆:重庆大学,2017.
[19]Carreras Villase?or H,Sánchez Arreguín J A,Herreraestrella A H.Trichoderma:sensing the environment for survival and dispersal[J].Microbiology,2012,158(1):3-16.
[20]Jie M,Wang L,Jiang X,et al.Functions of the C2H2 transcription factor gene thmea1 in Trichoderma harzianum under copper stress based on transcriptome analysis[J].BioMed Research International,2018,e8149682.
[21]Kuo J S,Patel M,Gamse J,et al.Opl:a zinc finger protein that regulates neural determination and patterning in xenopus[J].Development,1998,125(15):2867-2882.
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