靶向水稻脂氧合酶OsRCI-1的活性小分子化合物筛选体系的建立
|
摘要
为明确虫害诱导水稻脂氧合酶OsRCI-1的生化特性,通过构建水稻脂氧合酶OsRCI-1重组蛋白原核表达体系,对重组蛋白催化条件进行优化,并研究小分子化合物对OsRCI-1活性的影响。结果表明,缓冲液pH和反应温度都能影响水稻OsRCI-1蛋白的催化活性,当缓冲液pH为7.0时催化活性最佳,为169.9 mol·mg~(-1)·min~(-1);在检测的20~50℃反应温度范围内,在25℃和45℃时出现2个催化活性高峰,分别为68.2 mol·mg~(-1)·min~(-1)和70.4 mol·mg~(-1)·min~(-1)。利用建立的OsRCI-1活性检测体系筛选出4种小分子化合物,即水杨苷异羟肟酸、水杨酸、叔丁基对二苯酚和茉莉酸,均对水稻OsRCI-1活性具有调控作用,抑制中浓度依次为0.12、418.67、232.84、2 747.19μmol/L。表明本试验所建立的靶向水稻脂氧合酶活性化合物鉴定体系可用于活性小分子化合物的快速、高通量筛选。
In order to determine the biochemical characteristics of rice lipoxygenase Os RCI-1,a prokaryotic expression system of recombinant Os RCI-1 protein was established and the catalytic conditions of Os RCI-1 were optimized,and the effects of chemicals on the activity of Os RCI-1 were analyzed.The results showed that the p H of catalytic buffer and temperature could greatly affect the activity of OsRCI-1,with the highest catalytic activity(169.9 mol·mg~(-1)·min~(-1))observed at p H 7.0;in the range of20-50℃,the activity of Os RCI-1 showed two peaks(68.2 mol·mg~(-1)·min~(-1)and 70.4 mol·mg~(-1)·min~(-1))at25℃and 45℃,respectively.By using the Os RCI-1 activity detection system,four active chemicals including salicylhydroxamic acid,salicylic acid,tertiary butylhydroquinone,and jasmonic acid were identified,which could regulate Os RCI-1 activity,with a half inhibitory concentration(IC_(50))of 0.12,418.67,232.84,and 2 747.19μmol/L,respectively.To sum up,the screening system for Os RCI-1 activity was a quick and high-throughput system and could be used for identification of active chemicals against rice lipoxygenase.
In order to determine the biochemical characteristics of rice lipoxygenase Os RCI-1,a prokaryotic expression system of recombinant Os RCI-1 protein was established and the catalytic conditions of Os RCI-1 were optimized,and the effects of chemicals on the activity of Os RCI-1 were analyzed.The results showed that the p H of catalytic buffer and temperature could greatly affect the activity of OsRCI-1,with the highest catalytic activity(169.9 mol·mg~(-1)·min~(-1))observed at p H 7.0;in the range of20-50℃,the activity of Os RCI-1 showed two peaks(68.2 mol·mg~(-1)·min~(-1)and 70.4 mol·mg~(-1)·min~(-1))at25℃and 45℃,respectively.By using the Os RCI-1 activity detection system,four active chemicals including salicylhydroxamic acid,salicylic acid,tertiary butylhydroquinone,and jasmonic acid were identified,which could regulate Os RCI-1 activity,with a half inhibitory concentration(IC_(50))of 0.12,418.67,232.84,and 2 747.19μmol/L,respectively.To sum up,the screening system for Os RCI-1 activity was a quick and high-throughput system and could be used for identification of active chemicals against rice lipoxygenase.
引文
Christensen SA,Nemchenko A,Borrego E,Murray I,Sobhy IS,Boask L,De Blasio S,Erb M,Robert CAM,Vaughn KA,et al.2013.The maize lipoxygenase,Zm LOX10,mediates green leaf volatile,jasmonate and herbivore-induced plant volatile production for defense against insect attack.The Plant Journal,74(1):59-73
Christensen SA,Nemchenko A,Park YS,Borrego E,Huang PC,Schmelz EA,Kunze S,Feussne I,Yalpani N,Meeley R,et al.2014.The novel monocot-specific 9-lipoxygenase Zm LOX12 is required to mount and effective jasmonate-mediated defense against Fusarium verticillioides in maize.Molecular Plant-Microbe Interactions,27(11):1263-1276
Halitschke R,Baldwin IT.2003.Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata.Plant Journal,36(6):794-807
Huang JX,Cai MH,Long QZ,Liu LL,Lin QY,Jiang L,Chen SH,Wan JM.2014.Os LOX2,a rice type I lipoxygenase,confers opposite effects on seed germination and longevity.Transgenic Research,23(4):643-655
Ishiura S,Yoshimoto T,Villee CA.1986.Reticulocyte lipoxygenase,ingensin,and ATP-dependent proteolysis.FEBS Letters,201(1):87-93
Lou YG,Cheng JA.1997.Induced plant resistance to phytophagous insects.Acta Entomologica Sinica,40(3):320-331(in Chinese)[娄永根,程家安.1997.植物的诱导抗虫性.昆虫学报,40(3):320-331]
Montillet JL,Leonhardt N,Mondy S,Tranchimand S,Rumeau D,Boudsocq M,Garcia AV,Douki T,Bigeard J,Lauriere C,et al.2013.An abscisic acid-independent oxylipin pathway controls stomatal closure and immune defense in Arabidopsis.PLo S Biology,11(3):e1001513
Reverberi M,Fanelli C,Zjalic S,Briganti S,Picardo M,Ricelli A,Fabbri AA.2005.Relationship among lipoperoxides,jasmonates and indole-3-acetic acid formation in potato tuber after wounding.Free Radical Research,39(6):637-647
Reymond P,Farmer EE.1998.Jasmonate and salicylate as global signals for defense gene expression.Current Opinion in Plant Biology,1(5):404-411
Rouet-Mayer MA,Bureau JM,Lauriere C.1992.Identification and characterization of lipoxygenase isoforms in senescing carnation petals.Plant Physiology,98(3):971-978
Royo J,Leon J,Vancanneyt G,Albar JP,Rosahl S,Ortego F,Castanera P,Sanchez-Serrano JJ.1999.Antisense-mediated depletion of a potato lipoxygenase reduces wound induction of proteinase inhibitors and increases weight gain of insect pests.Proceedings of the National Academy of Sciences of the United States of America,96(3):1146-1151
Schaffrath U,Zabbai F,Dudler R.2000.Characterization of RCI-1,a chloroplastic rice lipoxygenase whose synthesis is induced by chemical plant resistance activators.European Journal of Biochemistry,267(19):5935-5942
Sun H.2013.Expression and purification of lipoxygenase and establishment of the inhibitor screening assay.Master Thesis.Guangzhou:Jinan University(in Chinese)[孙华.2013.脂氧合酶的表达纯化及抑制剂筛选体系的建立.硕士学位论文.广州:暨南大学]
Wang R,Shen WB,Liu LL,Jiang L,Liu YQ,Su N,Wan JM.2008.Anovel lipoxygenase gene from developing rice seeds confers dual position specificity and responds to wounding and insect attack.Plant Molecular Biology,66(4):401-414
Wasternack C.2015.How jasmonates earned their laurels:past and present.Journal of Plant Growth Regulation,34(4):761-794
Xin Z,Zhang Z,Chen Z,Sun X.2014.Salicylhydroxamic acid(SHAM)negatively mediates tea herbivore-induced direct and indirect defense against the tea geometrid Ectropis obliqua.Journal of Plant Research,127(4):565-572
Xu PS,Song J,Zhang ZY.2017.Prokaryotic expression of coat protein gene of Lily mottle virus and preparation,application of its antiserum.Journal of Plant Protection,44(4):630-635(in Chinese)[徐品三,宋炯,张郑瑶.2017.百合斑驳病毒外壳蛋白基因原核表达、抗血清制备及其应用.植物保护学报,44(4):630-635]
Yao FX,Zeng XC,Jiang HY,Fang JH,Wu XY.2006.Measurement of rice lipoxygenase activity by using linolenic acid as substrate.Acta Agriculturae Universitatis Jiangxiensis,28(2):183-186(in Chinese)[姚锋先,曾晓春,蒋海燕,方加海,吴晓玉.2006.水稻中以亚麻酸为底物的脂氧合酶活性测定.江西农业大学学报,28(2):183-186]
Yao ZL,Cao MJ,Wang X,Miao JS,Shen LZ,Deng JY,Wu HM,Xu ZH,Lou YG,Zhou GX.2014.Rice lipoxygenase gene Os RCI-1mediated pathway positively regulates resistance to rice striped stem borer.Journal of Environmental Entomology,36(4):507-515(in Chinese)[姚张良,曹梦娇,王霞,缪家顺,沈励泽,邓建宇,吴慧明,徐志宏,娄永根,周国鑫.2014.脂氧合酶Os RCI-1正调控水稻对二化螟的抗性.环境昆虫学报,36(4):507-515]
Zabbai F,Jarosch B,Schaffrath U.2004.Over-expression of chloroplastic lipoxygenase RCI1 cause PR1 transcript accumulation in transiently transformed rice.Physiological&Molecular Plant Pathology,64(1):37-43
Zhang JN,Hou JP,Weng XC.2009.Antioxidant activity of synthetic antioxidants and their oxidation products.Cereals and Oils Processing,(1):59-63(in Chinese)[章佳妮,侯建平,翁新楚.2009.合成抗氧化剂及其氧化产物的抗氧化活性.粮油加工,(1):59-63]
Zhou GX,Qi JF,Ren N,Cheng JA,Erb M,Mao BZ,Lou YG.2009.Silencing Os HI-LOX makes rice more susceptible to chewing herbivores,but enhances resistance to a phloem feeder.The Plant Journal,60(4):638-648
Christensen SA,Nemchenko A,Park YS,Borrego E,Huang PC,Schmelz EA,Kunze S,Feussne I,Yalpani N,Meeley R,et al.2014.The novel monocot-specific 9-lipoxygenase Zm LOX12 is required to mount and effective jasmonate-mediated defense against Fusarium verticillioides in maize.Molecular Plant-Microbe Interactions,27(11):1263-1276
Halitschke R,Baldwin IT.2003.Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata.Plant Journal,36(6):794-807
Huang JX,Cai MH,Long QZ,Liu LL,Lin QY,Jiang L,Chen SH,Wan JM.2014.Os LOX2,a rice type I lipoxygenase,confers opposite effects on seed germination and longevity.Transgenic Research,23(4):643-655
Ishiura S,Yoshimoto T,Villee CA.1986.Reticulocyte lipoxygenase,ingensin,and ATP-dependent proteolysis.FEBS Letters,201(1):87-93
Lou YG,Cheng JA.1997.Induced plant resistance to phytophagous insects.Acta Entomologica Sinica,40(3):320-331(in Chinese)[娄永根,程家安.1997.植物的诱导抗虫性.昆虫学报,40(3):320-331]
Montillet JL,Leonhardt N,Mondy S,Tranchimand S,Rumeau D,Boudsocq M,Garcia AV,Douki T,Bigeard J,Lauriere C,et al.2013.An abscisic acid-independent oxylipin pathway controls stomatal closure and immune defense in Arabidopsis.PLo S Biology,11(3):e1001513
Reverberi M,Fanelli C,Zjalic S,Briganti S,Picardo M,Ricelli A,Fabbri AA.2005.Relationship among lipoperoxides,jasmonates and indole-3-acetic acid formation in potato tuber after wounding.Free Radical Research,39(6):637-647
Reymond P,Farmer EE.1998.Jasmonate and salicylate as global signals for defense gene expression.Current Opinion in Plant Biology,1(5):404-411
Rouet-Mayer MA,Bureau JM,Lauriere C.1992.Identification and characterization of lipoxygenase isoforms in senescing carnation petals.Plant Physiology,98(3):971-978
Royo J,Leon J,Vancanneyt G,Albar JP,Rosahl S,Ortego F,Castanera P,Sanchez-Serrano JJ.1999.Antisense-mediated depletion of a potato lipoxygenase reduces wound induction of proteinase inhibitors and increases weight gain of insect pests.Proceedings of the National Academy of Sciences of the United States of America,96(3):1146-1151
Schaffrath U,Zabbai F,Dudler R.2000.Characterization of RCI-1,a chloroplastic rice lipoxygenase whose synthesis is induced by chemical plant resistance activators.European Journal of Biochemistry,267(19):5935-5942
Sun H.2013.Expression and purification of lipoxygenase and establishment of the inhibitor screening assay.Master Thesis.Guangzhou:Jinan University(in Chinese)[孙华.2013.脂氧合酶的表达纯化及抑制剂筛选体系的建立.硕士学位论文.广州:暨南大学]
Wang R,Shen WB,Liu LL,Jiang L,Liu YQ,Su N,Wan JM.2008.Anovel lipoxygenase gene from developing rice seeds confers dual position specificity and responds to wounding and insect attack.Plant Molecular Biology,66(4):401-414
Wasternack C.2015.How jasmonates earned their laurels:past and present.Journal of Plant Growth Regulation,34(4):761-794
Xin Z,Zhang Z,Chen Z,Sun X.2014.Salicylhydroxamic acid(SHAM)negatively mediates tea herbivore-induced direct and indirect defense against the tea geometrid Ectropis obliqua.Journal of Plant Research,127(4):565-572
Xu PS,Song J,Zhang ZY.2017.Prokaryotic expression of coat protein gene of Lily mottle virus and preparation,application of its antiserum.Journal of Plant Protection,44(4):630-635(in Chinese)[徐品三,宋炯,张郑瑶.2017.百合斑驳病毒外壳蛋白基因原核表达、抗血清制备及其应用.植物保护学报,44(4):630-635]
Yao FX,Zeng XC,Jiang HY,Fang JH,Wu XY.2006.Measurement of rice lipoxygenase activity by using linolenic acid as substrate.Acta Agriculturae Universitatis Jiangxiensis,28(2):183-186(in Chinese)[姚锋先,曾晓春,蒋海燕,方加海,吴晓玉.2006.水稻中以亚麻酸为底物的脂氧合酶活性测定.江西农业大学学报,28(2):183-186]
Yao ZL,Cao MJ,Wang X,Miao JS,Shen LZ,Deng JY,Wu HM,Xu ZH,Lou YG,Zhou GX.2014.Rice lipoxygenase gene Os RCI-1mediated pathway positively regulates resistance to rice striped stem borer.Journal of Environmental Entomology,36(4):507-515(in Chinese)[姚张良,曹梦娇,王霞,缪家顺,沈励泽,邓建宇,吴慧明,徐志宏,娄永根,周国鑫.2014.脂氧合酶Os RCI-1正调控水稻对二化螟的抗性.环境昆虫学报,36(4):507-515]
Zabbai F,Jarosch B,Schaffrath U.2004.Over-expression of chloroplastic lipoxygenase RCI1 cause PR1 transcript accumulation in transiently transformed rice.Physiological&Molecular Plant Pathology,64(1):37-43
Zhang JN,Hou JP,Weng XC.2009.Antioxidant activity of synthetic antioxidants and their oxidation products.Cereals and Oils Processing,(1):59-63(in Chinese)[章佳妮,侯建平,翁新楚.2009.合成抗氧化剂及其氧化产物的抗氧化活性.粮油加工,(1):59-63]
Zhou GX,Qi JF,Ren N,Cheng JA,Erb M,Mao BZ,Lou YG.2009.Silencing Os HI-LOX makes rice more susceptible to chewing herbivores,but enhances resistance to a phloem feeder.The Plant Journal,60(4):638-648