转基因ABP9玉米株系的耐盐性分析
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摘要
旨为分析转基因ABP9玉米的耐盐性,并进一步研究玉米耐盐的分子调控机制。通过对转基因ABP9玉米植株进行PCR、Southern blot和qRT-PCR分析鉴定;采用Hoagland营养液水培法,对转ABP9基因玉米及非转基因对照进行NaCl胁迫处理,分析二者在幼苗期的生理指标和基因表达差异。结果表明,转入的ABP9以单拷贝整合到基因组中,且能较高表达。与非转基因对照相比,转基因玉米幼苗NaCl胁迫下的叶绿素含量、Fv/Fm、渗透调节物质和抗氧化酶活性,以及盐胁迫后恢复过程中的叶片相对含水量均显著提高;而其丙二醛含量、相对电导率均显著降低。转录组测序和qRT-PCR分析显示,一系列盐胁迫应答基因在转基因玉米植株中上调表达。转入ABP9的增强表达提高了转基因玉米的耐盐能力。
This work is to analyze the salt tolerance of ABP9 transgenic maize,and to study the molecular regulation mechanism of salt tolerance in maize. ABP9 transgenic plants were identified by PCR,Southern blot and quantitative Reverse Transcription-PCR(qRT-PCR),and their responses at seedling stage to NaCl treatments in Hoagland nutrient solution were molecularly and physiologically characterized with non-transgenic plants as control. The results showed that the ABP9 transgene was integrated into the genome in a single copy and expressed at a higher level. Compared with the non-transgenic control,chlorophyll content,Fv/Fm,osmotic adjustment substance,and antioxidant enzyme activities in transgenic maize seedlings under NaCl stress,and the relative water content of leaves during recovery after salt stress all significantly increased,while the aldehyde content and relative conductivity significantly reduced. Several salt stress responsive genes were upregulated in transgenic plants revealed by transcriptome profiling and qRT-PCR analysis. Our results indicate that enhanced ABP9 expression level contributes to salt tolerance in transgenic maize,providing a valuable germplasm for maize salt tolerance breeding.
This work is to analyze the salt tolerance of ABP9 transgenic maize,and to study the molecular regulation mechanism of salt tolerance in maize. ABP9 transgenic plants were identified by PCR,Southern blot and quantitative Reverse Transcription-PCR(qRT-PCR),and their responses at seedling stage to NaCl treatments in Hoagland nutrient solution were molecularly and physiologically characterized with non-transgenic plants as control. The results showed that the ABP9 transgene was integrated into the genome in a single copy and expressed at a higher level. Compared with the non-transgenic control,chlorophyll content,Fv/Fm,osmotic adjustment substance,and antioxidant enzyme activities in transgenic maize seedlings under NaCl stress,and the relative water content of leaves during recovery after salt stress all significantly increased,while the aldehyde content and relative conductivity significantly reduced. Several salt stress responsive genes were upregulated in transgenic plants revealed by transcriptome profiling and qRT-PCR analysis. Our results indicate that enhanced ABP9 expression level contributes to salt tolerance in transgenic maize,providing a valuable germplasm for maize salt tolerance breeding.
引文
[1]Zorb C, Herbst R, Forreiter CEA. Short-term effects of salt exposure on the maize chloroplast protein pattern[J]. Proteomics, 2009, 9(17):4209-4220.
[2]张金林,李惠茹,郭姝媛,等.高等植物适应盐逆境研究进展[J].草业学报, 2015(12):220-236.
[3]李倩,王帅,隋颂扬,等.农杆菌介导的ZmRLK基因转化玉米的分析[J].分子植物育种, 2017(1):150-154.
[4]Soares A, Geilfus CM, Carpentier SC. Genotype-specific growth and proteomic responses of maize toward salt stress[J]. Front Plant Sci, 2018, 9:661.
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[6]Lindemose S, O’Shea C, Jensen MK, et al. Structure, function and networks of transcription factors involved in abiotic stress responses[J]. Int J Mol Sci, 2013, 14(3):5842-5878.
[7]SornarajP,SukanyaLSLM.Basicleucinezipper(bZIP)transcription factors involved in abiotic stresses A molecular model of a wheat bZIP factor and implications of its structure in function[J]. Biochimica et Biophysica Acta(BBA)-General Subjects 2016, 1860(1):46-56.
[8]John G. Guan LQ, Polidoros, et al. Catalases in plants:Gene structure, properties, regulation, and expression[J]. Oxidative Stress and the Molecular Biology of Antioxidant Defenses, 1997:343-406.
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[10]王昌陵,赵军,李英慧,等.转录因子ABP9转化大豆(Glycine max L.)及遗传转化条件优化[J].中国农业科学, 2008, 41(7):1908-1916.
[11]Zhang X, Wollenweber B, Jiang D, et al. Water deficits and heat shock effects on photosynthesis of a transgenic Arabidopsis thaliana constitutively expressing ABP9, a bZIP transcription factor[J]. J Exp Bot, 2008, 59(4):839-848.
[12]张磊,吴金霞,董芳,等.抗逆转ABP9基因黑麦草和高羊茅植株的鉴定[J].草业科学, 2010, 27(7):72-77.
[13]Zhang X, Wang L, Meng H, et al. Maize ABP9 enhances tolerance to multiple stresses in transgenic Arabidopsis by modulating ABA signaling and cellular levels of reactive oxygen species[J]. Plant Mol Biol, 2011, 75(4/5):365-378.
[14]Wang C, Lu G, Hao Y, et al. ABP9, a maize bZIP transcription factor, enhances tolerance to salt and drought in transgenic cotton[J]. Planta, 2017, 246(3):453-469.
[15]邹维华. ABP9启动子克隆、功能鉴定和编码区序列转录后6个核苷酸插入修饰分析[D].北京:中国农业科学院研究生院,2008.
[16]张宪政.植物叶绿素含量测定——丙酮乙醇混合液法[J].辽宁农业科学, 1986, 3(3):26-28.
[17]王立丰,王纪坤.叶绿素荧光动力学原理及在热带作物研究中的应用[J].热带农业科学, 2013, 33(11):16-23.
[18]Yadava P, Abhishek A, Singh R, et al. Advances in maize transformation technologies and development of transgenic maize[J]. Front Plant Sci, 2016, 7(421):1949.
[19]Kumar M, Choi J, An G, et al. Ectopic expression of OsSta2enhances salt stress tolerance in rice[J]. Front Plant Sci, 2017, 8:316.
[20]Lescano CI, Martini C, Gonzalez CA, et al. Allantoin accumulation mediated by allantoinase downregulation and transport by Ureide Permease 5 confers salt stress tolerance to Arabidopsis plants[J].Plant Mol Biol, 2016, 91(4/5):581-595.
[21]Mittal S, Kumari N, Sharma V. Differential response of salt stress on Brassica juncea:photosynthetic performance, pigment, proline,D1 and antioxidant enzymes[J]. Plant Physiol Biochem, 2012,54:17-26.
[22]Morant-Manceau A, Pradier E, Tremblin G. Osmotic adjustment,gas exchanges and chlorophyll fluorescence of a hexaploid triticale and its parental species under salt stress[J]. J Plant Physiol,2004, 161(1):25-33.
[23]Mehta P, Jajoo A, Mathur S, et al. Chlorophyll a fluorescence study revealing effects of high salt stress on Photosystem II in wheat leaves[J]. Plant Physiol Biochem, 2010, 48(1):16-20.
[24]Hajlaoui H, Ayeb N, Garrec JP, et al. Differential effects of salt stress on osmotic adjustment and solutes allocation on the basis of root and leaf tissue senescence of two silage maize(Zea mays L.)varieties[J]. Industrial Crops and Products, 2010, 31:122-130.
[25]Horie T, Karahara I, Katsuhara M. Salinity tolerance mechanisms in glycophytes:An overview with the central focus on rice plants[J]. Rice, 2012.
[26]Kandoi D, Mohanty S, Tripathy BC. Overexpression of plastidic maizeNADP-malatedehydrogenase(ZmNADP-MDH)in Arabidopsis thaliana confers tolerance to salt stress[J].Protoplasma, 2018, 255(2):547-563.
[27]Vijayalakshmi T, Vijayakumar AS, Kiranmai K, et al. Salt stress induced modulations in growth, compatible solutes and antioxidant enzymes response in two cultivars of safflower(Carthamus tinctorius L. Cultivar TSF1 and Cultivar SM)differing in salt tolerance[J]. American Journal of Plant Sciences, 2016:1802-1819.
[28]Farhangi-Abriz S, Torabian S. Antioxidant enzyme and osmotic adjustment changes in bean seedlings as affected by biochar under salt stress[J]. Ecotoxicol Environ Saf, 2017, 137:64-70.
[29]Ashraf M, Foolad MR. Roles of glycine betaine and proline in improving plant abiotic stress resistance[J]. Environmental and Experimental Botany, 2007, 59:206-216.
[30]Silveira G, Viégas R, Rocha M, etal. Proline accumulation and glutamine synthetase activity are increased bysalt-induced proteolysis in cashew leaves[J]. Plant Physiol, 2003, 160:115-123.
[31]Tripathi P, Rabara RC, Rushton PJ. A systems biology perspective on the role of WRKY transcription factors in drought responses in plants[J]. Planta, 2014, 239(2):255-266.
[32]Ning P, Liu C, Kang J, et al. Genome-wide analysis of WRKY transcription factors in wheat(Triticum aestivum L.)and differential expression under water deficit condition[J]. Peer J,2017, 5:e3232.
[33]Chen H, Lai Z, Shi J, et al. Roles of arabidopsis WRKY18,WRKY40 and WRKY60 transcription factors in plant responses to abscisic acid and abiotic stress[J]. BMC Plant Biol, 2010, 10:281.
[34]Pandey SP, Somssich IE. The role of WRKY transcription factors in plant immunity[J]. Plant Physiol, 2009, 150(4):1648-1655.
[35]Wang W, Vinocur B, Shoseyov O, et al. Role of plant heatshock proteins and molecular chaperones in the abiotic stress response[J]. Trends Plant Sci, 2004, 9(5):244-252.
[36]Waters ER. The molecular evolution of the small heat-shock proteins in plants[J]. Genetics, 1995, 141(2):785-795.
[37]Swindell WR, Huebner M, Weber AP. Transcriptional profiling of Arabidopsis heat shock proteins and transcription factors reveals extensive overlap between heat and non-heat stress response pathways[J]. BMC Genomics, 2007, 8:125.
[38]Chen JH, Jiang HW, Hsieh EJ, et al. Drought and salt stress tolerance of an Arabidopsis glutathione S-transferase U17 knockout mutant are attributed to the combined effect of glutathione and abscisic acid[J]. Plant Physiol, 2012, 158(1):340-351.
[39]Maxwell DP, Wang Y, McIntosh L. The alternative oxidase lowers mitochondrial reactive oxygen production in plant cells[J]. Proc Natl Acad Sci USA, 1999, 96(14):8271-8276.
[2]张金林,李惠茹,郭姝媛,等.高等植物适应盐逆境研究进展[J].草业学报, 2015(12):220-236.
[3]李倩,王帅,隋颂扬,等.农杆菌介导的ZmRLK基因转化玉米的分析[J].分子植物育种, 2017(1):150-154.
[4]Soares A, Geilfus CM, Carpentier SC. Genotype-specific growth and proteomic responses of maize toward salt stress[J]. Front Plant Sci, 2018, 9:661.
[5]Yamaguchi T, Blumwald E. Developing salt-tolerant crop plants:challenges and opportunities[J]. Trends Plant Sci, 2005, 10(12):615-620.
[6]Lindemose S, O’Shea C, Jensen MK, et al. Structure, function and networks of transcription factors involved in abiotic stress responses[J]. Int J Mol Sci, 2013, 14(3):5842-5878.
[7]SornarajP,SukanyaLSLM.Basicleucinezipper(bZIP)transcription factors involved in abiotic stresses A molecular model of a wheat bZIP factor and implications of its structure in function[J]. Biochimica et Biophysica Acta(BBA)-General Subjects 2016, 1860(1):46-56.
[8]John G. Guan LQ, Polidoros, et al. Catalases in plants:Gene structure, properties, regulation, and expression[J]. Oxidative Stress and the Molecular Biology of Antioxidant Defenses, 1997:343-406.
[9]Wang L, Zhao J, Fang YL. Gene cloning and function analysis of ABP9 protein which specifically binds to ABRE2 motif of maize Cat1 gene[J]. Chinese Science Bulletin, 2002, 47(22):1871-1875.
[10]王昌陵,赵军,李英慧,等.转录因子ABP9转化大豆(Glycine max L.)及遗传转化条件优化[J].中国农业科学, 2008, 41(7):1908-1916.
[11]Zhang X, Wollenweber B, Jiang D, et al. Water deficits and heat shock effects on photosynthesis of a transgenic Arabidopsis thaliana constitutively expressing ABP9, a bZIP transcription factor[J]. J Exp Bot, 2008, 59(4):839-848.
[12]张磊,吴金霞,董芳,等.抗逆转ABP9基因黑麦草和高羊茅植株的鉴定[J].草业科学, 2010, 27(7):72-77.
[13]Zhang X, Wang L, Meng H, et al. Maize ABP9 enhances tolerance to multiple stresses in transgenic Arabidopsis by modulating ABA signaling and cellular levels of reactive oxygen species[J]. Plant Mol Biol, 2011, 75(4/5):365-378.
[14]Wang C, Lu G, Hao Y, et al. ABP9, a maize bZIP transcription factor, enhances tolerance to salt and drought in transgenic cotton[J]. Planta, 2017, 246(3):453-469.
[15]邹维华. ABP9启动子克隆、功能鉴定和编码区序列转录后6个核苷酸插入修饰分析[D].北京:中国农业科学院研究生院,2008.
[16]张宪政.植物叶绿素含量测定——丙酮乙醇混合液法[J].辽宁农业科学, 1986, 3(3):26-28.
[17]王立丰,王纪坤.叶绿素荧光动力学原理及在热带作物研究中的应用[J].热带农业科学, 2013, 33(11):16-23.
[18]Yadava P, Abhishek A, Singh R, et al. Advances in maize transformation technologies and development of transgenic maize[J]. Front Plant Sci, 2016, 7(421):1949.
[19]Kumar M, Choi J, An G, et al. Ectopic expression of OsSta2enhances salt stress tolerance in rice[J]. Front Plant Sci, 2017, 8:316.
[20]Lescano CI, Martini C, Gonzalez CA, et al. Allantoin accumulation mediated by allantoinase downregulation and transport by Ureide Permease 5 confers salt stress tolerance to Arabidopsis plants[J].Plant Mol Biol, 2016, 91(4/5):581-595.
[21]Mittal S, Kumari N, Sharma V. Differential response of salt stress on Brassica juncea:photosynthetic performance, pigment, proline,D1 and antioxidant enzymes[J]. Plant Physiol Biochem, 2012,54:17-26.
[22]Morant-Manceau A, Pradier E, Tremblin G. Osmotic adjustment,gas exchanges and chlorophyll fluorescence of a hexaploid triticale and its parental species under salt stress[J]. J Plant Physiol,2004, 161(1):25-33.
[23]Mehta P, Jajoo A, Mathur S, et al. Chlorophyll a fluorescence study revealing effects of high salt stress on Photosystem II in wheat leaves[J]. Plant Physiol Biochem, 2010, 48(1):16-20.
[24]Hajlaoui H, Ayeb N, Garrec JP, et al. Differential effects of salt stress on osmotic adjustment and solutes allocation on the basis of root and leaf tissue senescence of two silage maize(Zea mays L.)varieties[J]. Industrial Crops and Products, 2010, 31:122-130.
[25]Horie T, Karahara I, Katsuhara M. Salinity tolerance mechanisms in glycophytes:An overview with the central focus on rice plants[J]. Rice, 2012.
[26]Kandoi D, Mohanty S, Tripathy BC. Overexpression of plastidic maizeNADP-malatedehydrogenase(ZmNADP-MDH)in Arabidopsis thaliana confers tolerance to salt stress[J].Protoplasma, 2018, 255(2):547-563.
[27]Vijayalakshmi T, Vijayakumar AS, Kiranmai K, et al. Salt stress induced modulations in growth, compatible solutes and antioxidant enzymes response in two cultivars of safflower(Carthamus tinctorius L. Cultivar TSF1 and Cultivar SM)differing in salt tolerance[J]. American Journal of Plant Sciences, 2016:1802-1819.
[28]Farhangi-Abriz S, Torabian S. Antioxidant enzyme and osmotic adjustment changes in bean seedlings as affected by biochar under salt stress[J]. Ecotoxicol Environ Saf, 2017, 137:64-70.
[29]Ashraf M, Foolad MR. Roles of glycine betaine and proline in improving plant abiotic stress resistance[J]. Environmental and Experimental Botany, 2007, 59:206-216.
[30]Silveira G, Viégas R, Rocha M, etal. Proline accumulation and glutamine synthetase activity are increased bysalt-induced proteolysis in cashew leaves[J]. Plant Physiol, 2003, 160:115-123.
[31]Tripathi P, Rabara RC, Rushton PJ. A systems biology perspective on the role of WRKY transcription factors in drought responses in plants[J]. Planta, 2014, 239(2):255-266.
[32]Ning P, Liu C, Kang J, et al. Genome-wide analysis of WRKY transcription factors in wheat(Triticum aestivum L.)and differential expression under water deficit condition[J]. Peer J,2017, 5:e3232.
[33]Chen H, Lai Z, Shi J, et al. Roles of arabidopsis WRKY18,WRKY40 and WRKY60 transcription factors in plant responses to abscisic acid and abiotic stress[J]. BMC Plant Biol, 2010, 10:281.
[34]Pandey SP, Somssich IE. The role of WRKY transcription factors in plant immunity[J]. Plant Physiol, 2009, 150(4):1648-1655.
[35]Wang W, Vinocur B, Shoseyov O, et al. Role of plant heatshock proteins and molecular chaperones in the abiotic stress response[J]. Trends Plant Sci, 2004, 9(5):244-252.
[36]Waters ER. The molecular evolution of the small heat-shock proteins in plants[J]. Genetics, 1995, 141(2):785-795.
[37]Swindell WR, Huebner M, Weber AP. Transcriptional profiling of Arabidopsis heat shock proteins and transcription factors reveals extensive overlap between heat and non-heat stress response pathways[J]. BMC Genomics, 2007, 8:125.
[38]Chen JH, Jiang HW, Hsieh EJ, et al. Drought and salt stress tolerance of an Arabidopsis glutathione S-transferase U17 knockout mutant are attributed to the combined effect of glutathione and abscisic acid[J]. Plant Physiol, 2012, 158(1):340-351.
[39]Maxwell DP, Wang Y, McIntosh L. The alternative oxidase lowers mitochondrial reactive oxygen production in plant cells[J]. Proc Natl Acad Sci USA, 1999, 96(14):8271-8276.