京水菜种子SU1抗菌蛋白的纯化、稳定性和抑菌机制
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摘要
为拓展抗菌蛋白在新型生物农药上的潜在应用价值,通过SP-Sepharose阳离子交换层析、Mone S阳离子交换层析和Superdex 75凝胶过滤层析从京水菜Brassica juncea var. multisecta种子中纯化得到一种分子量约为30 kD的SU1抗菌蛋白,采用纸片扩散法测定该抗菌蛋白的稳定性和抑菌活性,并采用荧光染色法分析其抗菌机制。结果表明,在pH为1～3和11～13条件下,SU1抗菌蛋白抑菌活性不受影响;40～80℃热处理和100 mmol/L Mn~(2+)、Al~(3+)、Zn~(2+)、Cu~(2+)、Fe3+、Pb~(2+)、Mg~(2+)、K~+和Cr~(3+)离子处理后,该抗菌蛋白仍具有抑菌活性,而100 mmol/L Ca~(2+)离子处理后该抑菌蛋白的抑菌活性消失。此外,乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)溶液处理后SU1抗菌蛋白的抑菌活性消失,再次加入Cr~(3+)离子时,其抑菌活性恢复。SU1抗菌蛋白能抑制7种植物病原真菌的生长,可破坏尖孢镰刀菌Fusarium oxysporum菌丝细胞膜透性和细胞核的完整性,并引起线粒体膜电势增加和菌丝细胞脱氧核糖核酸的分解。
In order to increase the potential application value of antifungal proteins as new bio-pesticides, a 30-kD antifungal protein SU1 was purified from Brassica juncea var. multisecta seeds with cation exchange chromatography on SP-Sepharose, cation exchange chromatography on Mono S and gel filtration chromatography on Superdex 75 in this study. The stability and antifungal activity were measured by using filter paper method and antifungal mechanism of the protein SU1 was studied by using fluorescent staining method. The results showed that the antifungal activity of protein SU1 was fully preserved in the pH range of 1-3 and 11-13, temperature range of 40-80℃, and 100 mmol/L Mn~(2+),Al~(3+), Zn~(2+), Cu~(2+), Fe~(3+), Pb~(2+), Mg~(2+), K~+ and Cr~(3+) ions. In contrast, the antifungal activity of protein SU1 disappeared following exposure to 100 mmol/L Ca~(2+)ions. In addition, its antifungal activity disappeared after treatment with ethylene diamine tetraacetic acid(EDTA) solution, and its antifungal activity was reinstituted following exposure to Cr~(3+) ions again. The antifungal protein SU1 inhibited the growth of seven species of phytopathogenic fungi, permeabilized fungal membrane and disrupted nuclear integrity,disrupted fungal mitochondrial transmembrane potential and upregulated deoxyribonucleic acid(DNA)degradation in Fusarium oxysporum hyphae.
In order to increase the potential application value of antifungal proteins as new bio-pesticides, a 30-kD antifungal protein SU1 was purified from Brassica juncea var. multisecta seeds with cation exchange chromatography on SP-Sepharose, cation exchange chromatography on Mono S and gel filtration chromatography on Superdex 75 in this study. The stability and antifungal activity were measured by using filter paper method and antifungal mechanism of the protein SU1 was studied by using fluorescent staining method. The results showed that the antifungal activity of protein SU1 was fully preserved in the pH range of 1-3 and 11-13, temperature range of 40-80℃, and 100 mmol/L Mn~(2+),Al~(3+), Zn~(2+), Cu~(2+), Fe~(3+), Pb~(2+), Mg~(2+), K~+ and Cr~(3+) ions. In contrast, the antifungal activity of protein SU1 disappeared following exposure to 100 mmol/L Ca~(2+)ions. In addition, its antifungal activity disappeared after treatment with ethylene diamine tetraacetic acid(EDTA) solution, and its antifungal activity was reinstituted following exposure to Cr~(3+) ions again. The antifungal protein SU1 inhibited the growth of seven species of phytopathogenic fungi, permeabilized fungal membrane and disrupted nuclear integrity,disrupted fungal mitochondrial transmembrane potential and upregulated deoxyribonucleic acid(DNA)degradation in Fusarium oxysporum hyphae.
引文
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Chu KT,Ng TB.2004.First report of a glutamine-rich antifungal peptide with immunomodulatory and antiproliferative activities from family Amaryllidaceae.Biochemical&Biophysical Research Communications,325(1):167-173
Fei W,Li H,Ma LY,Pei C,Kong XH.2015.Structure characteristics and antibacterial activities of histone-derived antibacterial peptides in aquatic animal.Journal of Henan Agricultural Sciences,44(4):9-13(in Chinese)[费威,李慧,马龙洋,裴超,孔祥会.2015.水生动物组蛋白衍生抗菌肽的结构特征和抗菌活性研究进展.河南农业科学,44(4):9-13]
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Lam YW,Wang HX,Ng TB.2000.A robust cysteine-deficient chitinase-like antifungal protein from inner shoots of the edible chive Allium tuberosum.Biochemical and Biophysical Research Communications,279(1):74-80
Lin P,Ng TB.2008.A novel and exploitable antifungal peptide from kale(Brassica alboglabra)seeds.Peptides,29(10):1664-1671
Lin P,Wong JH,Xia LX,Ng TB.2009.Campesin,a thermostable antifungal peptide with highly potent antipathogenic activities.Journal of Bioscience&Bioengineering,108(3):259-265
Liu XM,Cao AC,Wang QX,Yan DD,Ouyang CB,Li Y.2018.Current situation of bio-pesticide registration,extension and application in China.Plant Protection,44(5):101-107(in Chinese)[刘晓漫,曹坳程,王秋霞,颜冬冬,欧阳灿彬,李园.2018.我国生物农药的登记及推广应用现状.植物保护,44(5):101-107]
Ma XY.2011.Synthesis and properties of metal complexes of EDTAand DTPA derivatives of 6-aminopenicillanic acid.Master Thesis.Guangzhou:Guangdong University of Technology(in Chinese)[马肖依.2011.EDTA、DTPA氨基青霉烷酸衍生物的合成及其金属配合物性能研究.硕士学位论文.广州:广东工业大学]
Moreno AB,Del Pozo AM,Borja M,Segundo BS.2003.Activity of the antifungal protein from Aspergillus giganteus against Botrytis cinerea.Phytopathology,93(11):1344-1353
Qi DF,Zou LP,Zhou DB,Feng RJ,Gao ZF,Zhang XY.2017.Isolation,identification of strain GA1-2 and its antifungal activity against Fusarium oxysporum f.sp.cubense.Plant Protection,44(5):809-816(in Chinese)[起登凤,邹良平,周登博,冯仁军,高祝芬,张锡炎.2017.GA1-2菌株的分离鉴定及其对香蕉尖孢镰刀菌的抑菌效果.植物保护学报,44(5):809-816]
Sun H,Shi JL.2010.Antifungal characteristics of Bacillus subtilis CCTCC M207209 against Penicillium expansum:extraction,stability,and application of the antifungal substances in the liquid culture.Journal of Northwest A&F University(Natural Science Edition),38(1):201-208(in Chinese)[孙卉,师俊玲.2010.枯草芽孢杆菌CCTCC M207209抗扩展青霉特性研究--活性物质的提取、稳定性与应用.西北农林科技大学学报(自然科学版),38(1):201-208]
Tomar PPS,Nikhil K,Singh A,Selvakumar P,Roy P,Sharma AK.2014.Characterization of anticancer,DNase and antifungal activity of pumpkin 2S albumin.Biochemical and Biophysical Research Communications,448(4):349-354
Wang HX,Ng TB.2001.Isolation of a novel deoxyribonuclease with antifungal activity from Asparagus officinalis seeds.Biochemical and Biophysical Research Communications,289(1):120-124
Wang HX,Ng TB.2002.Isolation of an antifungal thaumatin-like protein from kiwi fruits.Phytochemistry,61(1):1-6
Wang HX,Ng TB.2004.Eryngin,a novel antifungal peptide from fruiting bodies of the edible mushroom Pleurotus eryngii.Peptides,25(1):1-5
Wang ZX,Wang YP,Zheng L,Yang XN,Liu HX,Guo JH.2014.Isolation and characterization of an antifungal protein from Bacillus licheniformis HS10.Biochemical and Biophysical Research Communications,454(1):48-52
Yan J,Yuan SS,Jiang LL,Ye XJ,Ng TB,Wu ZJ.2015.Plant antifungal proteins and their applications in agriculture.Applied Microbiology and Biotechnology,99(12):4961-4981
Ye XJ,Ng TB,Wu ZJ,Xie LH,Fang EF,Wong JH,Pan WL,Wing SS,Zhang YB.2011.Protein from red cabbage(Brassica oleracea)seeds with antifungal,antibacterial,and anticancer activities.Journal of Agricultural and Food Chemistry,59(18):10232-10238
Yuan SS,Qin WS,Zhang WW,Wang CC,Ye XJ.2018.Purification of antifungal protein from Brassica campestris L.seeds and analysis of antifungal activity and stability.Journal of Henan Agricultural Sciences,47(10):69-74(in Chinese)[袁素素,秦武洒,张玮玮,王财成,叶秀娟.2018.紫菜薹种子抗菌蛋白的分离纯化及其抑菌活性和稳定性研究.河南农业科学,47(10):69-74]
Zhang XX,Xie MC,Nan BD,Shuai NN,Qin YT.2015.Effects of different sowing date and planting density on the growth and yield of Brassica juncea var.multisecta L.H.Bailey.Modern Agricultural Science and Technology,(1):105-106(in Chinese)[张晓霞,颉敏昌,南炳东,帅娜娜,秦一统.2015.不同播期与种植密度对京水菜生长及产量的影响.现代农业科技,(1):105-106]
Zhao Y,Miao ZY,Li Y,Bai YJ.2014.Research progress on controlling against tomato gray mold.China Plant Protection,34(7):21-29(in Chinese)[赵杨,苗则彦,李颖,白元俊.2014.番茄灰霉病防治研究进展.中国植保导刊,34(7):21-29]
Zhong QP,Xie JJ,She SW.2002.Stabilities of antimicrobial protein produced by Bacillus natto.China Food Additives,(4):42-45(in Chinese)[钟青萍,谢俊杰,佘世望.2002.纳豆菌抗菌蛋白的稳定性.中国食品添加剂,(4):42-45]
Zhou R,Wang Y,Ren JJ,Wen SZ,Lai LM.2010.Effects of photoperiod and GA treatment on floral buds differentiation of water cabbage(Brassica japonica Sieb).Seed,29(6):75-77(in Chinese)[周荣,王艳,任吉君,文素珍,赖李明.2010.光周期和GA处理对京水菜花芽分化的影响.种子,29(6):75-77]
Zhu YM,Li S,Dong WJ,Jiang JP,Zhu BC.2018.Optimization of fermentation conditions for production of extracellular antibacterial protein by anti-diarrhea probiotics strain T-70-1.Heilongjiang Animal Science and Veterinary Medicine,(13):1-7(in Chinese)[朱永明,李森,董伟洁,姜军坡,朱宝成.2018.抗腹泻益生菌T-70-1胞外产抗菌蛋白发酵条件的优化.黑龙江畜牧兽医,(13):1-7]
Chan YS,Ng TB.2013.Northeast red beans produce a thermostable and pH-stable defensin-like peptide with potent antifungal activity.Cell Biochemistry and Biophysics,66(3):637-648
Chu KT,Ng TB.2004.First report of a glutamine-rich antifungal peptide with immunomodulatory and antiproliferative activities from family Amaryllidaceae.Biochemical&Biophysical Research Communications,325(1):167-173
Fei W,Li H,Ma LY,Pei C,Kong XH.2015.Structure characteristics and antibacterial activities of histone-derived antibacterial peptides in aquatic animal.Journal of Henan Agricultural Sciences,44(4):9-13(in Chinese)[费威,李慧,马龙洋,裴超,孔祥会.2015.水生动物组蛋白衍生抗菌肽的结构特征和抗菌活性研究进展.河南农业科学,44(4):9-13]
Ghosh P,Roy A,Hess D,Ghosh A,Das S.2015.Deciphering the mode of action of a mutant Allium sativum leaf agglutinin(mASAL),a potent antifungal protein on Rhizoctonia solani.BMC Microbiology,15:537
Gozia O,Ciopraga J,Bentia T,Lungu M,Zamfirescu I,Tudor R,Roseanu A,Nitu F.1993.Antifungal properties of lectin and new chitinases from potato tubers.Comptes Rendus de L'Academie des Sciences:Serie III,Sciences de la vie,316(8):788-792
Huang X,Xie WJ,Gong ZZ.2000.Characteristics and antifungal activity of a chitin binding protein from Ginkgo biloba.FEBS Letters,478(1/2):123-126
Lam YW,Wang HX,Ng TB.2000.A robust cysteine-deficient chitinase-like antifungal protein from inner shoots of the edible chive Allium tuberosum.Biochemical and Biophysical Research Communications,279(1):74-80
Lin P,Ng TB.2008.A novel and exploitable antifungal peptide from kale(Brassica alboglabra)seeds.Peptides,29(10):1664-1671
Lin P,Wong JH,Xia LX,Ng TB.2009.Campesin,a thermostable antifungal peptide with highly potent antipathogenic activities.Journal of Bioscience&Bioengineering,108(3):259-265
Liu XM,Cao AC,Wang QX,Yan DD,Ouyang CB,Li Y.2018.Current situation of bio-pesticide registration,extension and application in China.Plant Protection,44(5):101-107(in Chinese)[刘晓漫,曹坳程,王秋霞,颜冬冬,欧阳灿彬,李园.2018.我国生物农药的登记及推广应用现状.植物保护,44(5):101-107]
Ma XY.2011.Synthesis and properties of metal complexes of EDTAand DTPA derivatives of 6-aminopenicillanic acid.Master Thesis.Guangzhou:Guangdong University of Technology(in Chinese)[马肖依.2011.EDTA、DTPA氨基青霉烷酸衍生物的合成及其金属配合物性能研究.硕士学位论文.广州:广东工业大学]
Moreno AB,Del Pozo AM,Borja M,Segundo BS.2003.Activity of the antifungal protein from Aspergillus giganteus against Botrytis cinerea.Phytopathology,93(11):1344-1353
Qi DF,Zou LP,Zhou DB,Feng RJ,Gao ZF,Zhang XY.2017.Isolation,identification of strain GA1-2 and its antifungal activity against Fusarium oxysporum f.sp.cubense.Plant Protection,44(5):809-816(in Chinese)[起登凤,邹良平,周登博,冯仁军,高祝芬,张锡炎.2017.GA1-2菌株的分离鉴定及其对香蕉尖孢镰刀菌的抑菌效果.植物保护学报,44(5):809-816]
Sun H,Shi JL.2010.Antifungal characteristics of Bacillus subtilis CCTCC M207209 against Penicillium expansum:extraction,stability,and application of the antifungal substances in the liquid culture.Journal of Northwest A&F University(Natural Science Edition),38(1):201-208(in Chinese)[孙卉,师俊玲.2010.枯草芽孢杆菌CCTCC M207209抗扩展青霉特性研究--活性物质的提取、稳定性与应用.西北农林科技大学学报(自然科学版),38(1):201-208]
Tomar PPS,Nikhil K,Singh A,Selvakumar P,Roy P,Sharma AK.2014.Characterization of anticancer,DNase and antifungal activity of pumpkin 2S albumin.Biochemical and Biophysical Research Communications,448(4):349-354
Wang HX,Ng TB.2001.Isolation of a novel deoxyribonuclease with antifungal activity from Asparagus officinalis seeds.Biochemical and Biophysical Research Communications,289(1):120-124
Wang HX,Ng TB.2002.Isolation of an antifungal thaumatin-like protein from kiwi fruits.Phytochemistry,61(1):1-6
Wang HX,Ng TB.2004.Eryngin,a novel antifungal peptide from fruiting bodies of the edible mushroom Pleurotus eryngii.Peptides,25(1):1-5
Wang ZX,Wang YP,Zheng L,Yang XN,Liu HX,Guo JH.2014.Isolation and characterization of an antifungal protein from Bacillus licheniformis HS10.Biochemical and Biophysical Research Communications,454(1):48-52
Yan J,Yuan SS,Jiang LL,Ye XJ,Ng TB,Wu ZJ.2015.Plant antifungal proteins and their applications in agriculture.Applied Microbiology and Biotechnology,99(12):4961-4981
Ye XJ,Ng TB,Wu ZJ,Xie LH,Fang EF,Wong JH,Pan WL,Wing SS,Zhang YB.2011.Protein from red cabbage(Brassica oleracea)seeds with antifungal,antibacterial,and anticancer activities.Journal of Agricultural and Food Chemistry,59(18):10232-10238
Yuan SS,Qin WS,Zhang WW,Wang CC,Ye XJ.2018.Purification of antifungal protein from Brassica campestris L.seeds and analysis of antifungal activity and stability.Journal of Henan Agricultural Sciences,47(10):69-74(in Chinese)[袁素素,秦武洒,张玮玮,王财成,叶秀娟.2018.紫菜薹种子抗菌蛋白的分离纯化及其抑菌活性和稳定性研究.河南农业科学,47(10):69-74]
Zhang XX,Xie MC,Nan BD,Shuai NN,Qin YT.2015.Effects of different sowing date and planting density on the growth and yield of Brassica juncea var.multisecta L.H.Bailey.Modern Agricultural Science and Technology,(1):105-106(in Chinese)[张晓霞,颉敏昌,南炳东,帅娜娜,秦一统.2015.不同播期与种植密度对京水菜生长及产量的影响.现代农业科技,(1):105-106]
Zhao Y,Miao ZY,Li Y,Bai YJ.2014.Research progress on controlling against tomato gray mold.China Plant Protection,34(7):21-29(in Chinese)[赵杨,苗则彦,李颖,白元俊.2014.番茄灰霉病防治研究进展.中国植保导刊,34(7):21-29]
Zhong QP,Xie JJ,She SW.2002.Stabilities of antimicrobial protein produced by Bacillus natto.China Food Additives,(4):42-45(in Chinese)[钟青萍,谢俊杰,佘世望.2002.纳豆菌抗菌蛋白的稳定性.中国食品添加剂,(4):42-45]
Zhou R,Wang Y,Ren JJ,Wen SZ,Lai LM.2010.Effects of photoperiod and GA treatment on floral buds differentiation of water cabbage(Brassica japonica Sieb).Seed,29(6):75-77(in Chinese)[周荣,王艳,任吉君,文素珍,赖李明.2010.光周期和GA处理对京水菜花芽分化的影响.种子,29(6):75-77]
Zhu YM,Li S,Dong WJ,Jiang JP,Zhu BC.2018.Optimization of fermentation conditions for production of extracellular antibacterial protein by anti-diarrhea probiotics strain T-70-1.Heilongjiang Animal Science and Veterinary Medicine,(13):1-7(in Chinese)[朱永明,李森,董伟洁,姜军坡,朱宝成.2018.抗腹泻益生菌T-70-1胞外产抗菌蛋白发酵条件的优化.黑龙江畜牧兽医,(13):1-7]