龙须菜(Gracilariopsis lemaneiformis)中两种分支酸代谢酶对温度和水杨酸的响应及其原核表达研究
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摘要
龙须菜(Gracilariopsislemaneiformis)在我国主要用作鲍饵料和琼胶生产原料,但是南方夏季高温限制了龙须菜的栽培和产业化。抗逆植物激素水杨酸(salicylicacid,SA)与分支酸代谢之间存在着密切的联系。本文以两种分支酸代谢酶——异分支酸合成酶(isochorismate synthase,ICS)和分支酸变位酶(chorismatemutase,CM)为研究对象,分析了23℃(常温)和33℃(高温)条件下添加SA对龙须菜中两种分支酸代谢酶编码基因转录水平的影响。结果表明高温和SA不同程度地刺激了两种分支酸代谢酶转录水平的表达,如在3—12h,SA添加后ics表达量分别提高到常温对照组的2.84—4.76倍和高温对照组的1.25—1.62倍;在24h时,cm表达量分别提高到常温对照组的2.73倍和高温对照组的1.82倍。然后,我们将两种酶的编码基因分别转化到原核表达载体pET28a中,结果得到了多以包涵体形式大量表达的重组ICS和CM蛋白,其最佳诱导条件为0.1mmol/LIPTG在16℃诱导24h,并用镍柱初步纯化了重组蛋白。该研究为阐明温度和外源SA对龙须菜中两种分支酸代谢酶的影响规律,以及为从蛋白水平研究这两种代谢酶提供了资料。
The economic seaweed Gracilariopsis lemaneiformis is widely cultivated for feeding abalone and producing agar in China.However,high temperatures have limited its growth and industrialization in the southern coast.The stress-resistant phytohormone salicylic acid(SA) is intimately related to chorismate metabolism.In this study,the transcriptional level of two chorismate metabolic enzymes,isochorismate synthase(ICS) and chorismate mutase(CM) from G.lemaneiformis,were used to analyze the effects of the addition of SA under two temperature conditions.Results showed that high temperature(33℃) and SA stimulated the transcriptional expression of the two enzymes,e.g.,compared to the corresponding control,ics expression level ranged from 2.84 to 4.76 times at 23℃ and from 1.25 to 1.62 times at 33℃ after addition of SA within 3—12 h;cm expression level increased to 2.73 times at 23℃ and 1.82 times at 33℃ at 24 h.Then,the encoding genes of the two enzymes were transferred into pET28 a vector,respectively,and the recombinant ICS and CM proteins were expressed largely in the inclusion bodies.The optimal induction conditions of the recombinant proteins were at 16℃ for 24 h by 0.1 mmol/L IPTG.Finally,the recombinant proteins were preliminarily purified with a nickel column.This work provides the base for the research on the regulation of temperature and exogenous SA on the chorismate metabolism enzymes,and for the further research on the two enzymes at the protein levels in G.lemaneiformis.
The economic seaweed Gracilariopsis lemaneiformis is widely cultivated for feeding abalone and producing agar in China.However,high temperatures have limited its growth and industrialization in the southern coast.The stress-resistant phytohormone salicylic acid(SA) is intimately related to chorismate metabolism.In this study,the transcriptional level of two chorismate metabolic enzymes,isochorismate synthase(ICS) and chorismate mutase(CM) from G.lemaneiformis,were used to analyze the effects of the addition of SA under two temperature conditions.Results showed that high temperature(33℃) and SA stimulated the transcriptional expression of the two enzymes,e.g.,compared to the corresponding control,ics expression level ranged from 2.84 to 4.76 times at 23℃ and from 1.25 to 1.62 times at 33℃ after addition of SA within 3—12 h;cm expression level increased to 2.73 times at 23℃ and 1.82 times at 33℃ at 24 h.Then,the encoding genes of the two enzymes were transferred into pET28 a vector,respectively,and the recombinant ICS and CM proteins were expressed largely in the inclusion bodies.The optimal induction conditions of the recombinant proteins were at 16℃ for 24 h by 0.1 mmol/L IPTG.Finally,the recombinant proteins were preliminarily purified with a nickel column.This work provides the base for the research on the regulation of temperature and exogenous SA on the chorismate metabolism enzymes,and for the further research on the two enzymes at the protein levels in G.lemaneiformis.
引文
马长青,柏素花,董超华等,2016.苹果异分支酸合酶基因MdICS1的克隆与表达分析.植物生理学报,52(9):1361-1370
张亚兰,2013.海带磷酸甘露糖异构酶基因(PMI)的克隆、分析及表达.青岛:中国海洋大学硕士学位论文
曹福亮,欧祖兰,2008.水杨酸对银杏幼苗抗高温胁迫能力的影响.浙江林学院学报,25(6):756-759
彭琦,高建芹,周晓婴等,2016.甘蓝型油菜异分支酸合酶基因的克隆及信号通路相关基因的诱导表达.中国油料作物学报,38(1):7-12
Allert M,Cox J C,Hellinga H W,2010.Multifactorial determinants of protein expression in prokaryotic open reading frames.Journal of Molecular Biology,402(5):905-918
Catinot J,Buchala A,Abou-Mansour E et al,2008.Salicylic acid production in response to biotic and abiotic stress depends on isochorismate in Nicotiana benthamiana.FEBS Letters,582(4):473-478
Chen Z X,Zheng Z Y,Huang J L et al,2009.Biosynthesis of salicylic acid in plants.Plant Signaling and Behavior,4(6):493-496
Djamei A,Schipper K,Rabe F et al,2011.Metabolic priming by a secreted fungal effector.Nature,478(7369):395-398
Fei X G,2004.Solving the coastal eutrophication problem by large scale seaweed cultivation.Hydrobiologia,512(1-3):145-151
Hayat Q,Hayat S,Irfan M et al,2010.Effect of exogenous salicylic acid under changing environment:a review.Environmental and Experimental Botany,68(1):14-25
Hong T S,Roslan H A,2008.Cloning of a near complete Isochorismate synthase(ICS)cDNA from Morinda citrifolia L.Pertanika Journal of Tropical Agricultural Science,31(1):101-105
Hou S N,Lin L C,Lv Y et al,2018.Responses of lipoxygenase,jasmonic acid,and salicylic acid to temperature and exogenous phytohormone treatments in Gracilariopsis lemaneiformis(Rhodophyta).Journal of Applied Phycology,30:1-8,doi:10.1007/s10811-018-1514-2
Khow O,Suntrarachun S,2012.Strategies for production of active eukaryotic proteins in bacterial expression system.Asian Pacific Journal of Tropical Biomedicine,2(2):159-162
Liu S C,Chai X J,Guo W H et al,2014.Cloning,analysis and prokaryotic expression of DsSP gene from Dunaliella salina.Agricultural Science and Technology,15(6):907-915
Long H,Wang X,Xu J,2006.Molecular cloning and life-stage expression pattern of a new chorismate mutase gene from the root-knot nematode Meloidogyne arenaria.Plant Pathology,55(4):559-563
Maeda H,Dudareva N,2012.The shikimate pathway and aromatic amino acid biosynthesis in plants.Annual Review of Plant Biology,63:73-105
Muljono R A B,Talou J R,Scheffer J J C,2002.Biosynthesis of2,3-dihydroxybenzoic acid in transgenic Catharanthus roseus cell cultures overexpressing isochorismate synthase.Heterocycles,56(1-2):341-352
NovákováM,?a?ek V,Dobrev P I et al,2014.Plant hormones in defense response of Brassica napus to Sclerotinia sclerotiorum-reassessing the role of salicylic acid in the interaction with a necrotroph.Plant Physiology and Biochemistry,80:308-317
Nugroho L H,Verberne M C,Verpoorte R,2002.Activities of enzymes involved in the phenylpropanoid pathway in constitutively salicylic acid-producing tobacco plants.Plant Physiology and Biochemistry,40:755-760
Ogawa D,Nakajima N,Sano T et al,2005.Salicylic acid accumulation under O3 exposure is regulated by ethylene in tobacco plants.Plant and Cell Physiology,46(7):1062-1072
Ogawa D,Nakajima N,Tamaoki M et al,2007.The isochorismate pathway is negatively regulated by salicylic acid signaling in O3-exposed Arabidopsis.Planta,226(5):1277-1285
Pan Q H,Zhan J C,Liu H T et al,2006.Salicylic acid synthesized by benzoic acid 2-hydroxylase participates in the development of thermotolerance in pea plants.Plant Science,171(2):226-233
Sadeghi M,Dehghan S,Fischer R et al,2013.Isolation and characterization of isochorismate synthase and cinnamate4-hydroxylase during salinity stress,wounding,and salicylic acid treatment in Carthamus tinctorius.Plant Signaling&Behavior,8(11):e27335
Shafiee F,Rabbani M,Jahanian-Najafabadi A,2017.Optimization of the expression of DT386-BR2 fusion protein in Escherichia coli using response surface methodology.Advanced Biomedical Research,6(1):22
Strawn M A,Marr S K,Inoue K et al,2007.Arabidopsis isochorismate synthase functional in pathogen-induced salicylate biosynthesis exhibits properties consistent with a role in diverse stress responses.Journal of Biological Chemistry,282(8):5919-5933
Suzuki H,Takashima Y,Ishiguri F et al,2014.Proteomic analysis of responsive proteins induced in Japanese birch plantlet treated with salicylic acid.Proteomes,2(3):323-340
Wang L J,Chen S J,Kong W F et al,2006.Salicylic acid pretreatment alleviates chilling injury and affects the antioxidant system and heat shock proteins of peaches during cold storage.Postharvest Biology and Technology,41(3):244-251
Wang L Y,Zhang Y,Fu X Q et al,2017.Molecular cloning,characterization,and promoter analysis of the isochorismate synthase(AaICS1)gene from Artemisia annua.Journal of Zhejiang University-Science B,18(8):662-673
Yuan Y N,Chung J D,Fu X Y et al,2009.Alternative splicing and gene duplication differentially shaped the regulation of isochorismate synthase in Populus and Arabidopsis.Proceedings of the National Academy of Sciences of the United States of America,106(51):22020-22025
张亚兰,2013.海带磷酸甘露糖异构酶基因(PMI)的克隆、分析及表达.青岛:中国海洋大学硕士学位论文
曹福亮,欧祖兰,2008.水杨酸对银杏幼苗抗高温胁迫能力的影响.浙江林学院学报,25(6):756-759
彭琦,高建芹,周晓婴等,2016.甘蓝型油菜异分支酸合酶基因的克隆及信号通路相关基因的诱导表达.中国油料作物学报,38(1):7-12
Allert M,Cox J C,Hellinga H W,2010.Multifactorial determinants of protein expression in prokaryotic open reading frames.Journal of Molecular Biology,402(5):905-918
Catinot J,Buchala A,Abou-Mansour E et al,2008.Salicylic acid production in response to biotic and abiotic stress depends on isochorismate in Nicotiana benthamiana.FEBS Letters,582(4):473-478
Chen Z X,Zheng Z Y,Huang J L et al,2009.Biosynthesis of salicylic acid in plants.Plant Signaling and Behavior,4(6):493-496
Djamei A,Schipper K,Rabe F et al,2011.Metabolic priming by a secreted fungal effector.Nature,478(7369):395-398
Fei X G,2004.Solving the coastal eutrophication problem by large scale seaweed cultivation.Hydrobiologia,512(1-3):145-151
Hayat Q,Hayat S,Irfan M et al,2010.Effect of exogenous salicylic acid under changing environment:a review.Environmental and Experimental Botany,68(1):14-25
Hong T S,Roslan H A,2008.Cloning of a near complete Isochorismate synthase(ICS)cDNA from Morinda citrifolia L.Pertanika Journal of Tropical Agricultural Science,31(1):101-105
Hou S N,Lin L C,Lv Y et al,2018.Responses of lipoxygenase,jasmonic acid,and salicylic acid to temperature and exogenous phytohormone treatments in Gracilariopsis lemaneiformis(Rhodophyta).Journal of Applied Phycology,30:1-8,doi:10.1007/s10811-018-1514-2
Khow O,Suntrarachun S,2012.Strategies for production of active eukaryotic proteins in bacterial expression system.Asian Pacific Journal of Tropical Biomedicine,2(2):159-162
Liu S C,Chai X J,Guo W H et al,2014.Cloning,analysis and prokaryotic expression of DsSP gene from Dunaliella salina.Agricultural Science and Technology,15(6):907-915
Long H,Wang X,Xu J,2006.Molecular cloning and life-stage expression pattern of a new chorismate mutase gene from the root-knot nematode Meloidogyne arenaria.Plant Pathology,55(4):559-563
Maeda H,Dudareva N,2012.The shikimate pathway and aromatic amino acid biosynthesis in plants.Annual Review of Plant Biology,63:73-105
Muljono R A B,Talou J R,Scheffer J J C,2002.Biosynthesis of2,3-dihydroxybenzoic acid in transgenic Catharanthus roseus cell cultures overexpressing isochorismate synthase.Heterocycles,56(1-2):341-352
NovákováM,?a?ek V,Dobrev P I et al,2014.Plant hormones in defense response of Brassica napus to Sclerotinia sclerotiorum-reassessing the role of salicylic acid in the interaction with a necrotroph.Plant Physiology and Biochemistry,80:308-317
Nugroho L H,Verberne M C,Verpoorte R,2002.Activities of enzymes involved in the phenylpropanoid pathway in constitutively salicylic acid-producing tobacco plants.Plant Physiology and Biochemistry,40:755-760
Ogawa D,Nakajima N,Sano T et al,2005.Salicylic acid accumulation under O3 exposure is regulated by ethylene in tobacco plants.Plant and Cell Physiology,46(7):1062-1072
Ogawa D,Nakajima N,Tamaoki M et al,2007.The isochorismate pathway is negatively regulated by salicylic acid signaling in O3-exposed Arabidopsis.Planta,226(5):1277-1285
Pan Q H,Zhan J C,Liu H T et al,2006.Salicylic acid synthesized by benzoic acid 2-hydroxylase participates in the development of thermotolerance in pea plants.Plant Science,171(2):226-233
Sadeghi M,Dehghan S,Fischer R et al,2013.Isolation and characterization of isochorismate synthase and cinnamate4-hydroxylase during salinity stress,wounding,and salicylic acid treatment in Carthamus tinctorius.Plant Signaling&Behavior,8(11):e27335
Shafiee F,Rabbani M,Jahanian-Najafabadi A,2017.Optimization of the expression of DT386-BR2 fusion protein in Escherichia coli using response surface methodology.Advanced Biomedical Research,6(1):22
Strawn M A,Marr S K,Inoue K et al,2007.Arabidopsis isochorismate synthase functional in pathogen-induced salicylate biosynthesis exhibits properties consistent with a role in diverse stress responses.Journal of Biological Chemistry,282(8):5919-5933
Suzuki H,Takashima Y,Ishiguri F et al,2014.Proteomic analysis of responsive proteins induced in Japanese birch plantlet treated with salicylic acid.Proteomes,2(3):323-340
Wang L J,Chen S J,Kong W F et al,2006.Salicylic acid pretreatment alleviates chilling injury and affects the antioxidant system and heat shock proteins of peaches during cold storage.Postharvest Biology and Technology,41(3):244-251
Wang L Y,Zhang Y,Fu X Q et al,2017.Molecular cloning,characterization,and promoter analysis of the isochorismate synthase(AaICS1)gene from Artemisia annua.Journal of Zhejiang University-Science B,18(8):662-673
Yuan Y N,Chung J D,Fu X Y et al,2009.Alternative splicing and gene duplication differentially shaped the regulation of isochorismate synthase in Populus and Arabidopsis.Proceedings of the National Academy of Sciences of the United States of America,106(51):22020-22025