镉胁迫对灵芝菌丝体生长及代谢产物积累的影响
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摘要
目的研究镉(Cd)胁迫对灵芝菌丝生长及代谢产物积累的影响,探讨Cd胁迫影响生长和代谢产物积累的机制,为灵芝生产栽培过程中控制Cd元素提供依据。方法在Cd质量浓度分别为0、0.5、1.0、4.0、10.0、40.0 mg/L下培养灵芝菌丝体,对其生物量积累、胞内活性氧(ROS)水平、膜氧化损伤、抗氧化酶活性及ROS调控相关酶表达量进行分析。结果 Cd质量浓度达到4.0 mg/L时,抑制菌丝生长;胞内ROS水平、H2O2及丙二醛(MDA)含量显著上升,分别提升了76%、46%、325%,且随着Cd质量浓度的增加呈上升趋势;NADPH氧化酶基因(NOXA)、超氧化物歧化酶基因(SOD1,SOD4)、过氧化氢酶基因(CAT)表达量显著上调。Cd质量浓度达到10.0mg/L时抑制效果显著,菌落生长直径及发酵菌丝干质量抑制率分别为26.15%、32.78%,灵芝总三萜抑制率为33.7%,对总蛋白合成的抑制率为30.3%,对多糖抑制不显著。Cd质量浓度达到40.0 mg/L时,抗坏血酸过氧化物酶基因(APX)及谷胱甘肽过氧化物酶基因(GPX)表达量显著上调。随着Cd质量浓度的增加,SOD、CAT、APX、GPX酶活性都呈先上升后下降趋势,Cd质量浓度达到1.0mg/L时,GPX酶活性下降,APX酶活性上升显著。外源添加diphenyleneiodonium chloride(DPI)、N-乙酰-L-半胱氨酸(NAC)、维生素C(VC)对Cd胁迫灵芝胞内清除ROS水平及降低MDA含量作用显著。结论 Cd胁迫造成灵芝菌丝产量及代谢物积累下降,可能是由于Cd离子抑制GPX酶活性下降,造成H2O2积累,引起ROS水平上升及膜氧化损伤,抑制菌丝生长及代谢产物积累,同时调控NOXA基因表达量上调,造成抗氧化酶活性及表达量上升来提高机体对活性氧的清除。因此在灵芝的生产过程中要控制Cd元素质量浓度小于1 mg/L。
Objective To study the effects of cadmium stress on mycelial growth and accumulation of metabolites in Ganoderma lucidum, and to explore the mechanisms affecting growth and accumulation of metabolites, and to provide evidence for controlling cadmium in the production and cultivation of G. lucidum. Methods The mycelium of G. lucidum was cultured under the conditions of heavy metal ion cadmium concentration of 0, 0.5, 1, 4, 10, and 40 mg/L, and its biomass accumulation, intracellular ROS level, membrane oxidative damage, anti-oxidant enzyme activity, and ROS regulation related enzyme expression were analyzed. Results When the concentration of cadmium reached 4 mg/L, the mycelial growth was inhibited. The levels of intracellular ROS, H2 O2, and MDA increased significantly, increasing by 76%, 46% and 325%, respectively, and increased with the increase of cadmium concentration; The NADPH expression levels of oxidase gene(NOXA), superoxide dismutase gene(SOD1 and SOD4), and CATalase gene(CAT) were significantly up-regulated. When the cadmium concentration reached 10 mg/L, the inhibitory effect was significant. The colony growth diameter and the dry weight inhibition rate of fermentation mycelium were 26.15% and 32.78%, respectively. The total triterpenoid inhibition rate of G. lucidum was 33.7%, and the inhibition rate of total protein synthesis was 30.3%. Inhibition of polysaccharides was not significant. When the cadmium concentration reached 40 mg/L, the expression levels of Ascorbate peroxidase gene(APX) and Glutathione peroxidase gene(GPX) were significantly up-regulated. With the increase of cadmium concentration, the activities of SOD, CAT, APX, and GPX increased first and then decreased. When the concentration of cadmium reached 1 mg/L, the activity of GPX decreased and the activity of APX increased significantly. Exogenous addition of diphenyleneiodonium chloride(DPI), N-acetyl-L-cysteine(NAC) and vitamin C(VC) had significant effects on cadmium-induced G. lucidum clearance of ROS and reduction of MDA content. Conclusion Cadmium stress causes the decrease of mycelial production and metabolite accumulation of G. lucidum, which may be due to the inhibition of GPX activity by cadmium ions, resulting in the accumulation of H2 O2, causing the increase of ROS level and membrane oxidative damage, inhibiting mycelial growth and accumulation of metabolites, and regulating NOX. Up-regulation of gene expression results in an increase in anti-oxidant enzyme activity and expression to increase the clearance of reactive oxygen species. Therefore, the cadmium content should be controlled within the range of 1 mg/L during the production process.
Objective To study the effects of cadmium stress on mycelial growth and accumulation of metabolites in Ganoderma lucidum, and to explore the mechanisms affecting growth and accumulation of metabolites, and to provide evidence for controlling cadmium in the production and cultivation of G. lucidum. Methods The mycelium of G. lucidum was cultured under the conditions of heavy metal ion cadmium concentration of 0, 0.5, 1, 4, 10, and 40 mg/L, and its biomass accumulation, intracellular ROS level, membrane oxidative damage, anti-oxidant enzyme activity, and ROS regulation related enzyme expression were analyzed. Results When the concentration of cadmium reached 4 mg/L, the mycelial growth was inhibited. The levels of intracellular ROS, H2 O2, and MDA increased significantly, increasing by 76%, 46% and 325%, respectively, and increased with the increase of cadmium concentration; The NADPH expression levels of oxidase gene(NOXA), superoxide dismutase gene(SOD1 and SOD4), and CATalase gene(CAT) were significantly up-regulated. When the cadmium concentration reached 10 mg/L, the inhibitory effect was significant. The colony growth diameter and the dry weight inhibition rate of fermentation mycelium were 26.15% and 32.78%, respectively. The total triterpenoid inhibition rate of G. lucidum was 33.7%, and the inhibition rate of total protein synthesis was 30.3%. Inhibition of polysaccharides was not significant. When the cadmium concentration reached 40 mg/L, the expression levels of Ascorbate peroxidase gene(APX) and Glutathione peroxidase gene(GPX) were significantly up-regulated. With the increase of cadmium concentration, the activities of SOD, CAT, APX, and GPX increased first and then decreased. When the concentration of cadmium reached 1 mg/L, the activity of GPX decreased and the activity of APX increased significantly. Exogenous addition of diphenyleneiodonium chloride(DPI), N-acetyl-L-cysteine(NAC) and vitamin C(VC) had significant effects on cadmium-induced G. lucidum clearance of ROS and reduction of MDA content. Conclusion Cadmium stress causes the decrease of mycelial production and metabolite accumulation of G. lucidum, which may be due to the inhibition of GPX activity by cadmium ions, resulting in the accumulation of H2 O2, causing the increase of ROS level and membrane oxidative damage, inhibiting mycelial growth and accumulation of metabolites, and regulating NOX. Up-regulation of gene expression results in an increase in anti-oxidant enzyme activity and expression to increase the clearance of reactive oxygen species. Therefore, the cadmium content should be controlled within the range of 1 mg/L during the production process.
引文
[1]戴玉成,杨祝良.中国药用真菌名录及部分名称的修订[J].菌物学报, 2008, 27(6):801-8
[2]戴玉成.中国多孔茵名录机[J].菌物学报, 2009,28(3):315-
[3] Li Y Q, Wang S F. Antihepatitis B activities of ganoderic acidfrom Ganoderma lucidum[J]. Biotechnol Lett, 2006,28(11):837-
[4] W,individual ganoderic acids and expression of biosynthetic genes in liquid static and shaking cultures of Ganoderma lucidum[J]. Appl Microbiol Bioechnol, 2010, 87:457-466.
[5]董峰,吴琼,李向阳,等. ROS在镉诱导HK-2细胞氧化损伤和凋亡中的作用研究[J].中国细胞生物学学报, 2018, 40(10):1662-1669.
[6]赵连华,杨银慧,胡一晨,等.我国中药材中重金属污染现状分析及对策研究[J].中草药, 2014, 45(9):1199-1206.
[7] Baldantoni D, Morra L, Zaccardelli L, et al.Cadmiumaccumulation in leaves of leafy vegetables[J]. Ecotox Environ Safe, 2016, 123:89-94.
[8]张鼎.镉中毒及锌对镉中毒的作用机理研究[D].武汉:华中农业大学, 2015.
[9]张晓柠.灵芝对四种重金属富集作用的研究[D].北京:中国协和医科大学, 2007.
[10] Labuschagne C F, Brenkman A B. Current methods in quantifying ROS and oxidative damage in Caenorhabditis elegans and other model organism of aging[J]. Ageing Res Rev, 2013, 12(4):918-930.
[11] Wulf D. Free radicals in the physiological control of cell function[J]. Physiol Reviews, 2002, 82(1):47-95.
[12]董峰,吴琼,李向阳,等. ROS在镉诱导HK-2细胞氧化损伤和凋亡中的作用研究[J].中国细胞生物学学报, 2018, 40(10):1662-1669.
[13] Wang Y, Fang J, Leonard S S, et al. Cadmium inhibits the electron transfer chain and induces reactive oxygen species[J]. Free Rad Biol Med, 2004, 36:1434-1443.
[14] Thijssen S, Cuypers A, Maringwa J, et al. Low cadmium exposure triggers a biphasic oxidative stress response in mice kidneys[J]. Toxicology, 2007, 236(12):29-41.
[15]王松华,张华,崔元戎,等.镉对灵芝菌丝抗氧化系统的影响[J].应用生态学报, 2008(6):1355-1361.
[16]穆大帅.灵芝基因沉默体系的建立及NADPH氧化酶基因家族功能分析[D].南京:南京农业大学, 2013.
[17] Thijssen S, Cuypers A, Maringwa J, et al. Low cadmium exposure triggers a biphasic oxidative stress response in mice kidneys[J]. Toxicology, 2007, 236:29-41.
[18] Daud M K, He Q L, Lei M, et al. Ultrastructural,metabolic and proteomic changes in leaves of upland cotton in response to cadmium stress[J]. Chemosphere,2015, 120:309-320.
[19]颜鹏.重金属胁迫下废水中白腐真菌抗氧化应激的研究[D].无锡:江南大学2017.
[20]周丽,郎多勇,张文晋,等. NaCl胁迫对银柴胡生长及生理生化特性的影响[J].中草药, 2014, 45(19):2829-2833.
[21] Cuypers A, Plusquin M, Remans T, et al. Cadmium stress:An oxidative challenge[J]. Biometals, 2010, 23:927-940.
[22] Sato Y. Heat shock-mediated APX gene expression and protection against chilling injury in rice seedlings[J]. J Exper Bot, 2001, 52(354):145-151.
[23]刘娜,闫博,李涌泉,等.镉对长江华溪蟹谷胱甘肽系统的影响[J].环境科学, 2008(8):2302-2307.
[24]罗其勇.重金属暴露引起鱼体氧化应激反应的研究进展[J].安徽农业科学, 2018, 46(25):32-35.
[2]戴玉成.中国多孔茵名录机[J].菌物学报, 2009,28(3):315-
[3] Li Y Q, Wang S F. Antihepatitis B activities of ganoderic acidfrom Ganoderma lucidum[J]. Biotechnol Lett, 2006,28(11):837-
[4] W,individual ganoderic acids and expression of biosynthetic genes in liquid static and shaking cultures of Ganoderma lucidum[J]. Appl Microbiol Bioechnol, 2010, 87:457-466.
[5]董峰,吴琼,李向阳,等. ROS在镉诱导HK-2细胞氧化损伤和凋亡中的作用研究[J].中国细胞生物学学报, 2018, 40(10):1662-1669.
[6]赵连华,杨银慧,胡一晨,等.我国中药材中重金属污染现状分析及对策研究[J].中草药, 2014, 45(9):1199-1206.
[7] Baldantoni D, Morra L, Zaccardelli L, et al.Cadmiumaccumulation in leaves of leafy vegetables[J]. Ecotox Environ Safe, 2016, 123:89-94.
[8]张鼎.镉中毒及锌对镉中毒的作用机理研究[D].武汉:华中农业大学, 2015.
[9]张晓柠.灵芝对四种重金属富集作用的研究[D].北京:中国协和医科大学, 2007.
[10] Labuschagne C F, Brenkman A B. Current methods in quantifying ROS and oxidative damage in Caenorhabditis elegans and other model organism of aging[J]. Ageing Res Rev, 2013, 12(4):918-930.
[11] Wulf D. Free radicals in the physiological control of cell function[J]. Physiol Reviews, 2002, 82(1):47-95.
[12]董峰,吴琼,李向阳,等. ROS在镉诱导HK-2细胞氧化损伤和凋亡中的作用研究[J].中国细胞生物学学报, 2018, 40(10):1662-1669.
[13] Wang Y, Fang J, Leonard S S, et al. Cadmium inhibits the electron transfer chain and induces reactive oxygen species[J]. Free Rad Biol Med, 2004, 36:1434-1443.
[14] Thijssen S, Cuypers A, Maringwa J, et al. Low cadmium exposure triggers a biphasic oxidative stress response in mice kidneys[J]. Toxicology, 2007, 236(12):29-41.
[15]王松华,张华,崔元戎,等.镉对灵芝菌丝抗氧化系统的影响[J].应用生态学报, 2008(6):1355-1361.
[16]穆大帅.灵芝基因沉默体系的建立及NADPH氧化酶基因家族功能分析[D].南京:南京农业大学, 2013.
[17] Thijssen S, Cuypers A, Maringwa J, et al. Low cadmium exposure triggers a biphasic oxidative stress response in mice kidneys[J]. Toxicology, 2007, 236:29-41.
[18] Daud M K, He Q L, Lei M, et al. Ultrastructural,metabolic and proteomic changes in leaves of upland cotton in response to cadmium stress[J]. Chemosphere,2015, 120:309-320.
[19]颜鹏.重金属胁迫下废水中白腐真菌抗氧化应激的研究[D].无锡:江南大学2017.
[20]周丽,郎多勇,张文晋,等. NaCl胁迫对银柴胡生长及生理生化特性的影响[J].中草药, 2014, 45(19):2829-2833.
[21] Cuypers A, Plusquin M, Remans T, et al. Cadmium stress:An oxidative challenge[J]. Biometals, 2010, 23:927-940.
[22] Sato Y. Heat shock-mediated APX gene expression and protection against chilling injury in rice seedlings[J]. J Exper Bot, 2001, 52(354):145-151.
[23]刘娜,闫博,李涌泉,等.镉对长江华溪蟹谷胱甘肽系统的影响[J].环境科学, 2008(8):2302-2307.
[24]罗其勇.重金属暴露引起鱼体氧化应激反应的研究进展[J].安徽农业科学, 2018, 46(25):32-35.