高温胁迫下糙皮侧耳胞内钙离子对热激蛋白基因表达的调控
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摘要
以糙皮侧耳(Pleurotus ostreatus)菌株CCMSSC00389为材料,研究高温胁迫(40℃、120 min)下钙离子的响应及其对热激蛋白的调控。在高温胁迫条件下,在培养基中添加胞内钙离子螯合剂、胞外钙离子螯合剂、钙调蛋白抑制剂、质膜钙通道抑制剂、三磷酸肌醇受体拮抗剂,利用钙离子特异性荧光探针检测菌丝体胞内钙离子含量,采用荧光定量PCR检测热激蛋白的基因表达量。结果表明,高温胁迫促进胞内钙离子浓度升高,增加Hsp60、Grp78、Hsp90、Hsp104等4种热激蛋白的基因表达量;培养基中添加钙离子螯合剂或抑制剂会降低胞内钙离子浓度,抑制这些热激蛋白的基因表达。研究结果表明高温胁迫会诱导糙皮侧耳胞内钙离子浓度升高,参与信号转导,从而调控热激蛋白的表达来缓解高温胁迫引起的伤害。
The response of calcium ion(Ca~(2+))and its regulation on heat shock proteins(Hsps)in Pleurotus ostreatus(CCMSSC00389)under heat stress(40 ℃,120 min)were investigated in the study.Under heat stress,an intracellular Ca~(2+) chelator,an extracellular Ca~(2+) chelator,a calmodulin inhibitor,a plasma membrane Ca~(2+) channel inhibitor,and an antagonist of inositol 1,4,5-trisphosphate receptor were supplemented into the medium respectively,then the cytosolic Ca~(2+) was detected by a Ca~(2+) fluorescent probe,and the gene expression levels of Hsps were detected by fluorescence quantitative PCR.It was shown that heat stress induced the increase of cytosolic Ca~(2+) concentration and the gene expression of four kinds of Hsps(Hsp60,Grp78,Hsp90 and Hsp104),while the addition of the Ca~(2+) chelators or inhibitors in the medium decreased the cytosolic Ca~(2+) concentration and inhibited the gene expression of Hsps.The results revealed that cytosolic Ca~(2+) in P.ostreatus participated in signal transduction responsive to heat stress,thereby regulating the gene expression of Hsps to alleviate damages caused by heat stress.
The response of calcium ion(Ca~(2+))and its regulation on heat shock proteins(Hsps)in Pleurotus ostreatus(CCMSSC00389)under heat stress(40 ℃,120 min)were investigated in the study.Under heat stress,an intracellular Ca~(2+) chelator,an extracellular Ca~(2+) chelator,a calmodulin inhibitor,a plasma membrane Ca~(2+) channel inhibitor,and an antagonist of inositol 1,4,5-trisphosphate receptor were supplemented into the medium respectively,then the cytosolic Ca~(2+) was detected by a Ca~(2+) fluorescent probe,and the gene expression levels of Hsps were detected by fluorescence quantitative PCR.It was shown that heat stress induced the increase of cytosolic Ca~(2+) concentration and the gene expression of four kinds of Hsps(Hsp60,Grp78,Hsp90 and Hsp104),while the addition of the Ca~(2+) chelators or inhibitors in the medium decreased the cytosolic Ca~(2+) concentration and inhibited the gene expression of Hsps.The results revealed that cytosolic Ca~(2+) in P.ostreatus participated in signal transduction responsive to heat stress,thereby regulating the gene expression of Hsps to alleviate damages caused by heat stress.
引文
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[24]张哲,宋水山,边子睿,等.植物细胞中钙离子作用的研究进展[J].安黴农业科学,2011,39(24):14525-14527.
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[26]CHEUNG WY.Calmodulin plays a pivotal role in cellular regulation[J].Science,1980,207(4426):19-27.
[27]辛苗苗.高温胁迫下香菇疏水蛋白和热激蛋白的转录表达量变化及功能研究[D].南京:南京农业大学,2016.
[28]樊志和,周人纲,李晓芝,等.钙-钙调素与小麦苗中热激蛋白的诱导[J].植物生理学报,2000,26(4):331-336.
[29]刘宏涛,赵和,李冰,等.钙-钙调素对小麦hsp26基因表达的影响[J].植物学报,2001,43(7):766-768.
[30]FEDOSEEVA IV,VARAKINA NN,RUSALEVA TM,et al.Effect of calcium ions on the Hsp104 synthesis and heat tolerance of Saccharomyces cerevisiae[J].Microbiology,2010,79(2):153-159.
[2]ZHANG X,REN A,LI MJ,et al.Heat stress modulates mycelium growth,heat shock protein expression,Ganoderic acid biosynthesis and hyphal branching of Ganoderma lucidum via cytosolic Ca~(2+)[J].Applied and Environmental Microbiology,2016,82(14):4112-4115.
[3]宋驰.两种侧耳高温胁迫导致的细胞凋亡研究和球孢白僵菌胁迫响应转录机制的研究[D].北京:中国农业科学院,2015.
[4]孟利娟.一氧化氮和海藻糖对白灵侧耳高温响应抗氧化途径的影响研究[M].北京:中国农业科学院,2015.
[5]雷敏.热胁迫下糙皮侧耳菌丝体海藻糖代谢调控研究[D].北京:中国农业大学,2018.
[6]刘秀明.糙皮侧耳和肺形侧耳热胁迫响应的海藻糖代谢调控研究[D].北京:中国农业科学院,2013.
[7]WANG LN,WU XL,GAO W,et al.Differential expression patterns of Pleurotus ostreatus catalase genes during developmental stages and under heat stress[J].Genes,2017,8(11):335.
[8]曾后清,张亚仙,汪尚,等.植物钙/钙调素介导的信号转导系统[J].植物学报,2016,51(5):705-723.
[9]郭静,蒲咏梅,张东才.钙离子信号与细胞凋亡[J].生物物理学报,2005,21(1):1-18.
[10]唐海林,苏琦.钙离子信号与细胞凋亡关系的研究进展[J].南华大学学报(医学版),2006,34(5):663-666.
[11]LIAO CC,ZHENG Y,GUO Y.MYB30 transcription factor regulates oxidative and heat stress responses through ANNEXIN-mediated cytosolic calcium signaling in Arabidopsis[J].The New Phytologist,2017,216(1):163-177.
[12]CRAIG EA,GAMBILL BD,NELSON RJ.Heat shock proteins:molecular chaperones of protein biogenesis[J].Microbiological Reviews,1993,57(2):402-414.
[13]FRAGKOSTEFANAKIS S,ROTH S,SCHLEIFF E,et al.Prospects of engineering thermotolerance in crops through modulation of heat stress transcription factor and heat shock protein networks[J].Plant,Cell and Environment,2015,38(9):1881-1895.
[14]WANG WX,BASIA V,ODED S,et al.Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response[J].Trends in Plant Science,2004,9(5):244-252.
[15]ZOU YJ,ZHANG MJ,QU JB,et al.iTRAQ-Based Quantitative Proteomic Analysis Reveals Proteomic Changes in Mycelium of Pleurotus ostreatus in Response to Heat Stress and Subsequent Recovery[J].Frontiers in Microbiology,2018,9:2368.
[16]SAGE SO,RINK TJ.The kinetics of changes in intracellular calcium concentration in Fura-2-loaded human platelets[J].Journal of Biological Chemistry,1987,262(34):16364-16369.
[17]赵玉龙,李娜娜,赵飞云,等.Ca~(2+)/CaM参与了sHSP26增强玉米叶绿体耐干旱高温复合胁迫[J].河南农业大学学报,2016,50(4):447-452.
[18]GHOSH TK,EIS PS,MULLANEY JM,et al.Competitive,reversible,and potent antagonism of inositol 1,4,5-trisphosphate-activated calcium release by heparin[J].Journal of Biological Chemistry,1988,263(23):11075-11079.
[19]ZHANG T,CAO EH,LI JF.A laser scanning confocal microscopy method:simultaneous detection of intracellular Ca~(2+)and apoptosis using Fluo-3 and Hoechst 33342[J].Analytical and Quantitative Cytology and Histology,2000,22(2):93-97.
[20]LI Y,LIU S,ZHANG F,et al.Expression of the microRNAs hsa-miR-15a and hsa-miR-16-1 in lens epithelial cells of patients with age-related cataract[J].International Journal of Clinical and Experimental Medicine,2015,8(2):2405-2410.
[21]KNIGHT H.Calcium signaling during abiotic stress in plants[J].International Review of Cytology,1999,195:269-324.
[22]杨卫星.钙转导半夏高温信号及其生理响应[D].重庆:西南大学,2015.
[23]尚忠林,毛国红,孙大业.植物细胞内钙信号的特异性[J].植物生理学报,2003,39(2):93-100.
[24]张哲,宋水山,边子睿,等.植物细胞中钙离子作用的研究进展[J].安黴农业科学,2011,39(24):14525-14527.
[25]查仁明.植物细胞第二信使研究进展[J].渝西学院学报(自然科学版),2002,1(2):18-21.
[26]CHEUNG WY.Calmodulin plays a pivotal role in cellular regulation[J].Science,1980,207(4426):19-27.
[27]辛苗苗.高温胁迫下香菇疏水蛋白和热激蛋白的转录表达量变化及功能研究[D].南京:南京农业大学,2016.
[28]樊志和,周人纲,李晓芝,等.钙-钙调素与小麦苗中热激蛋白的诱导[J].植物生理学报,2000,26(4):331-336.
[29]刘宏涛,赵和,李冰,等.钙-钙调素对小麦hsp26基因表达的影响[J].植物学报,2001,43(7):766-768.
[30]FEDOSEEVA IV,VARAKINA NN,RUSALEVA TM,et al.Effect of calcium ions on the Hsp104 synthesis and heat tolerance of Saccharomyces cerevisiae[J].Microbiology,2010,79(2):153-159.