LaeA调控黑曲霉形态发展、赭曲霉毒素合成和氧化耐受
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摘要
探究LaeA蛋白对黑曲霉形态发展,赭曲霉毒素合成和氧化耐受的影响,为控制黑曲霉侵染和赭曲霉毒素合成提供理论基础。利用农杆菌介导的同源重组方法,构建黑曲霉laeA基因敲除菌株,比较野生株和敲除菌株在形态发展、赭曲霉毒素合成和氧化耐受性的差异。结果表明相比野生型菌株,△laeA菌株生长速度减慢,产孢减少,菌丝长且松散,枝节相对较少;赭曲霉毒素A(ochratoxin A,OTA)合成量大幅减少,光照培养7 d,OTA减少92.45%,黑暗培养7 d,OTA减少99.03%;对氧的敏感性增加,10 mmol/L H_2O_2可完全抑制△laeA菌株生长,5 mmol/L H_2O_2胁迫条件下,△laeA菌株相比于野生菌株,细胞内抗氧化酶活性显著降低。LaeA蛋白正调控黑曲霉产孢、OTA合成及氧化耐受性。
To explore the function of the LaeA protein with regard to morphological development,ochratoxin biosynthesis and oxidative stress tolerance in Aspergillus niger. Also,to contribute to a better understanding of the laeA regulatory mechanism in Aspergillus niger. We proposed the laeA gene as a potential target in developing a control strategy to prevent Aspergillus niger infection and ochratoxin biosynthesis. An Aspergillus niger laeA gene disruption strain was constructed by Agrobacterium-mediated homologous recombination. A wild type(WT)was compared with the △laeA strain to examine differences in morphological development,ochratoxin biosynthesis and oxidative stress tolerance. Compared with WT,the △laeA strain had reduced hyphal growth,hyphal branching,and conidia production. Moreover,the △laeA strain almost abolished the bios-ynthesis of ochratoxin A(OTA). After 7 d of growth in both light and dark conditions,OTA production decreased by 92.45 % and 99.03 %,respectively,when compared with WT. The △laeA strain also showed increased sensitivity to oxidative stress. When 10 mmol/L H_2O_2 was added to the growth medium,the △laeA strain could not grow,whereas the WT strain was able to form colonies. Compared with WT,the △laeA strain has significantly decreased antioxidant enzyme activity under 5 mmol/L H_2O_2 oxidative stress. The LaeA protein acts as a positive regulator on conidia production,OTA biosynthesis,and oxidative stress tolerance in Aspergillus niger.
To explore the function of the LaeA protein with regard to morphological development,ochratoxin biosynthesis and oxidative stress tolerance in Aspergillus niger. Also,to contribute to a better understanding of the laeA regulatory mechanism in Aspergillus niger. We proposed the laeA gene as a potential target in developing a control strategy to prevent Aspergillus niger infection and ochratoxin biosynthesis. An Aspergillus niger laeA gene disruption strain was constructed by Agrobacterium-mediated homologous recombination. A wild type(WT)was compared with the △laeA strain to examine differences in morphological development,ochratoxin biosynthesis and oxidative stress tolerance. Compared with WT,the △laeA strain had reduced hyphal growth,hyphal branching,and conidia production. Moreover,the △laeA strain almost abolished the bios-ynthesis of ochratoxin A(OTA). After 7 d of growth in both light and dark conditions,OTA production decreased by 92.45 % and 99.03 %,respectively,when compared with WT. The △laeA strain also showed increased sensitivity to oxidative stress. When 10 mmol/L H_2O_2 was added to the growth medium,the △laeA strain could not grow,whereas the WT strain was able to form colonies. Compared with WT,the △laeA strain has significantly decreased antioxidant enzyme activity under 5 mmol/L H_2O_2 oxidative stress. The LaeA protein acts as a positive regulator on conidia production,OTA biosynthesis,and oxidative stress tolerance in Aspergillus niger.
引文
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[19]曹张磊,王德培,张岚.提高根癌农杆菌介导黑曲霉转化效率的研究[J].天津科技大学学报,2016,31(2):20-25
[20]陈浩宇,徐瑞涛,程志翔,等.H2O2对黑曲霉氧化胁迫机理的研究[J].生物技术通报,2018,34(4):1-6
[21]KosalkováK,García-Estrada C,Ullán R V,et al.The global regulator LaeA controls penicillin biosynthesis,pigmentation and sporulation,but not roquefortine C synthesis in Penicillium chrysogenum[J].Biochimie,2009,91(2):214-225
[22]Crespo-Sempere A,Marín S,Sanchis V,et al.Ve A and LaeA transcriptional factors regulate ochratoxin A biosynthesis in Aspergillus carbonarius[J].International Journal of Food Microbiology,2013,166(3):479-486
[23]Wu D,Oide S,Zhang N,et al.ChLae1 and ChVel1 regulate T-toxin production,virulence,oxidative stress response,and development of the maize pathogen Cochliobolus heterostrophus[J].PLoS Pathogens,2012,8(2):e1002542
[2]Schuster E,Dunncoleman N,Frisvad J,et al.On the safety of Aspergillus niger-a review[J].Applied Microbiology Biotechnology,2002,59(4/5):426-435
[3]Lucchetta G,Bazzo I,Dal Cortivo G,et al.Occurrence of black Aspergilli and ochratoxin A on grapes in Italy[J].Toxins,2010,2(4):840-855
[4]Gautam A K,Sharma S,Avasthi S,et al.Diversity,pathogenicity and toxicology of A.niger:an important spoilage fungi[J].Research Journal of Microbiology,2011,6(3):270-280
[5]Frisvad J C,Petersen L M,Lyhne E K,et al.Formation of sclerotia and production of indoloterpenes by Aspergillus niger and other species in section Nigri[J].PLoS One,2014,9(4):e94857
[6]Geiser D M.Sexual structures in Aspergillus:morphology,importance and genomics[J].Medical Mycology,2009,47(1):S21-S26
[7]Liu X,Williams C E,Nemacheck J A,et al.Reactive oxygen species are involved in plant defense against a gall midge[J].Plant Physiology,2010,152:985-999
[8]El Khoury A,Atoui A.Ochratoxin A:general overview and actual molecular status[J].Toxins(Basel),2010,2(4):461-493
[9]Wang Y,Wang L,Liu F,et al.Ochratoxin A producing fungi,biosynthetic pathway and regulatory mechanisms[J].Toxins,2016,8(3):83
[10]Shwab E K,Keller N P.Regulation of secondary metabolite production in filamentous ascomycetes[J].Mycology Research,2008,112(2):225-230
[11]Bok J W,Keller N P.LaeA a regulator of secondary metabolism in Aspergillus spp.[J].Eukaryotic Cell,2004,3(2):527-535
[12]徐志超,孙超,徐江,等.Velvet和LaeA蛋白调控药用菌发育及其活性成分合成相关研析进展[J].药学学报,2014,49(11):1520-1527
[13]Reyes D Y,Bok J W,Berger H,et al.Heterochromatic marks are associated with the repression of secondary metabolism clusters in Aspergillus nidulans[J].Molecular Microbiology,2010,76(6):1376-1386
[14]Oda K,Kobayashi A,Ohashi S,et al.Aspergillus oryzae laeA regulator kojic acid synthesis genes[J].Bioscience,Biotechnology,and Biochemistry,2011,75(9):1832-1834
[15]Niu J,Arentshorst M,Nair P D,et al.Identification of a classical mutant in the industrial host Aspergillus niger by systems genetics:LaeA is required for citric acid production and regulates the formation of some secondary metabolites[J].Genes/Genomes/Genetics(Bethesda),2016,6(1):193-204
[16]Zhang J,Zhu L Y,Chen H Y,et al.A polyketide synthase encoded by the gene An15g07920 is involved in the biosynthesis of ochratoxin A in Aspergillus niger[J].Journal of Agricultural and Food Chemistry,2016,64(51):9680
[17]李载平.《分子克隆实验指南》(第3版)[J].科学通报,2002,47(24):1888
[18]林春花,郑服丛.稻瘟菌MgORP1基因敲除突变株的构建及其表型分析[J].微生物学报,2008,48(9):1160-1167
[19]曹张磊,王德培,张岚.提高根癌农杆菌介导黑曲霉转化效率的研究[J].天津科技大学学报,2016,31(2):20-25
[20]陈浩宇,徐瑞涛,程志翔,等.H2O2对黑曲霉氧化胁迫机理的研究[J].生物技术通报,2018,34(4):1-6
[21]KosalkováK,García-Estrada C,Ullán R V,et al.The global regulator LaeA controls penicillin biosynthesis,pigmentation and sporulation,but not roquefortine C synthesis in Penicillium chrysogenum[J].Biochimie,2009,91(2):214-225
[22]Crespo-Sempere A,Marín S,Sanchis V,et al.Ve A and LaeA transcriptional factors regulate ochratoxin A biosynthesis in Aspergillus carbonarius[J].International Journal of Food Microbiology,2013,166(3):479-486
[23]Wu D,Oide S,Zhang N,et al.ChLae1 and ChVel1 regulate T-toxin production,virulence,oxidative stress response,and development of the maize pathogen Cochliobolus heterostrophus[J].PLoS Pathogens,2012,8(2):e1002542