过量表达盐芥TsIPK2基因增强转基因水稻耐盐性
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摘要
[目的]本试验以野生型(WT)和转盐芥TsIPK2基因的水稻为材料,研究NaCl胁迫条件下过量表达TsIPK2基因对水稻植株抗盐胁迫能力的影响。[方法]取水稻材料种子和其3叶龄幼苗,分别在不同NaCl浓度(0、50、100、150、200mmol/L)下进行处理。检测WT与过量表达TsIPAK2基因水稻种子的发芽率、主根长和芽长、幼苗的丙二醛(MDA)和脯氨酸的含量,超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性,以及与胁迫相关的5个基因的表达。[结果]在盐胁迫下,与野生型相比,转基因水稻具有更好的发芽率、主根长和芽长。野生型和转基因水稻两者的脯氨酸含量增加,转基因水稻的积累量显著高于野生型,但是转基因水稻MDA含量增加幅度小于野生型。野生型和转基因水稻幼苗SOD酶活性均增加,但转基因植株的酶活性显著高于野生型;二者POD酶活性呈现先升高后下降的趋势,但是二者活性没有显著的差别;转基因水稻的CAT活性也呈现先升高后下降的趋势,然而野生型水稻的CAT活性在盐胁迫下没有显著的变化。高盐处理后,野生型和转基因水稻的5个与胁迫相关的基因表达倍数都增加,与野生型相比,转基因水稻的OsP5CS1、OsSOD、OsCATB和OsLEA3的表达量显著升高,而OsPOX1基因的表达量变化不显著。[结论]过量表达TsIPAK2基因能够通过增强水稻的渗透调节能力、抗氧化胁迫能力并调节胁迫相关基因的表达,以提高水稻的耐盐性。
[Objectives]In this study,the wild type(WT) and the transgenic rice carrying TsIPK2 gene were evaluated to clarify responsive characteristics of rice under salt stress.[Methods ]The seeds and 3-leaves-old seedlings of the transgenic rice and the WT were subjected to the solutions containing concentrations of NaCl 0,50, 100, 150, 200 mmol/L, and physiologically profiled by investigating the seed germination rate, main root and shoot length,MDA and proline content,SOD, POD and CAT activity, and the relative expression of target genes associated with salt stress.[Results ]The results showed that the germination rate,main root length and shoot height in the transgenic rice were significantly higher than those in the wild type. Although the accumulations of proline and MDA in the two types were increased, the MDA contents in transgenic type were lower than that in the wild type. While the accumulation of proline in the transgenic rice was significantly higher than that in the wild type under salt stress. Meanwhile, under the salt stress treatment, the activity of SOD in the transgenic rice was much higher than that in the wild type, but the POD activity increased firstly and then decreased without obvious differences in both the transgenic rice and the wild type, and the CAT activity demonstrated similar trends in both the transgenic rice and the wild type. High-salt stress induced up-regulation of five stress-responsive genes at transcriptional level, in which, the expressions of the OsP5 CS1, Os SOD, OsCATB and Os LEA 3 were significantly increased in transgenic rice compared with the wild type except for the OsPOX1. [Conclusions ]This study demonstrates that the overexpression of the TsIPK2 gene improved the salt tolerance in rice via participating in the regulations of compatible solutes, antioxidant capacity and expressions of the stress-responsive genes.
[Objectives]In this study,the wild type(WT) and the transgenic rice carrying TsIPK2 gene were evaluated to clarify responsive characteristics of rice under salt stress.[Methods ]The seeds and 3-leaves-old seedlings of the transgenic rice and the WT were subjected to the solutions containing concentrations of NaCl 0,50, 100, 150, 200 mmol/L, and physiologically profiled by investigating the seed germination rate, main root and shoot length,MDA and proline content,SOD, POD and CAT activity, and the relative expression of target genes associated with salt stress.[Results ]The results showed that the germination rate,main root length and shoot height in the transgenic rice were significantly higher than those in the wild type. Although the accumulations of proline and MDA in the two types were increased, the MDA contents in transgenic type were lower than that in the wild type. While the accumulation of proline in the transgenic rice was significantly higher than that in the wild type under salt stress. Meanwhile, under the salt stress treatment, the activity of SOD in the transgenic rice was much higher than that in the wild type, but the POD activity increased firstly and then decreased without obvious differences in both the transgenic rice and the wild type, and the CAT activity demonstrated similar trends in both the transgenic rice and the wild type. High-salt stress induced up-regulation of five stress-responsive genes at transcriptional level, in which, the expressions of the OsP5 CS1, Os SOD, OsCATB and Os LEA 3 were significantly increased in transgenic rice compared with the wild type except for the OsPOX1. [Conclusions ]This study demonstrates that the overexpression of the TsIPK2 gene improved the salt tolerance in rice via participating in the regulations of compatible solutes, antioxidant capacity and expressions of the stress-responsive genes.
引文
[1] Foti M,Audhya A,Emr S D. Sacl lipid phosphatase and Stt4phosphatidylinositol 4-kinase regulate a pool of phosphatidylinositol4-phosphate that functions in the control of the actin cytoskeleton and vacuole morphology[J]. Molecular Biology of the Cell, 2001, 12(8):2396-2411.
[2] Odom A R, Stahlberg A, Wente S R, et al. A role for nuclear inositol1, 4, 5-trisphosphate kinase in transcriptional control[J]. Science,2000, 287(5460):2026-2029.
[3] Nalaskowski M M, Deschermeier C, Fanick W, et al. The human homologue of yeast ArgRIII protein is an inositol phosphate multikinase with predominantly nuclear localization[J]. Biochemical Journal, 2002, 366(2):549-556.
[4] Frederick J P, Mattiske D, Wofford J A, et al. An essential role for an inositol polyphosphate multikinase, Ipk2, in mouse embryogenesis and second messenger production[J]. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(24):8454-8459.
[5] Stevensonpaulik J, Odom A R, York J D. Molecular and biochemical characterization of two plant inositol polyphosphate 6-/3-/5-kinases[J]. Journal of Biological Chemistry, 2002, 277(45):42711-42718.
[6] Xia H J, Brearley C, Elge S, et al. Arabidopsis inositol polyphosphate6-/3-kinase is a nuclear protein that complements a yeast mutant lacking a functional argr-mcml transcription complex[J]. Plant Cell,2003, 15(2):449-463.
[7] Suzuki M, Tanaka K, Kuwano M, et al. Expression pattern of inositol phosphate-related enzymes in rice(Oryza sativa L.):implications for the phytic acid biosynthetic pathway[J]. Gene, 2007, 405(1-2):55-64.
[8] Caddick S E K, Harrison C J, Stavridou I, et al. A Solanumtuberosum inositol phosphate kinase(StITPK1)displaying inositol phosphate-inositol phosphate and inositol phosphate-ADP phosphotransferase activities[J]. Febs Letters, 2008, 582(12):1731-1737.
[9] Zhu J Q, Zhang J T, Tang R J, et al. Molecular characterization of ThIPK2, an inositol polyphosphate kinase gene homolog from Thellungiella halophila, and its heterologous expression to improve abiotic stress tolerance in Brassica napus[J]. Physiologia Plantarum,2009, 136(4):407-425.
[10] Yang L, Tang R, Zhu J, et al. Enhancement of stress tolerance in transgenic tobacco plants constitutively expressing AtIpk2β, an inositol polyphosphate 6-/3-kinase from Arabidopsis thaliana[J].Plant Molecular Biology,2008, 66(4):329-343.
[11] Liu M, Li D, Wang Z, et al. Transgenic expression of ThIPK2 gene in soybean improves stress tolerance, oleic acid content and seed size[J].Plant Cell Tissue&Organ Culture, 2012, 111(3):277-289.
[12] Zhu J K. Plant salt tolerance[J]. Trends in Plant Science, 2001, 6(2):66-71.
[13] Sairam R K, Srivastava G C, Agarwal S, et al. Differences in antioxidant activity in response to salinity stress in tolerant and susceptible wheat genotypes[J]. Biologia Plantarum, 2005, 49(1):85-91.
[14] Singh M, Kumar J, Singh S, et al. Roles of osmoprotectants in improving salinity and drought tolerance in plants:a review[J].Reviews in Environmental Science&Bio-technology, 2015, 14(3):407-426.
[15] Mittler R. Oxidative stress, antioxidants and stress tolerance[J].Trends in Plant Science, 2002, 7(9):405-410.
[16] Negi N P. Overexpression of CuZnSOD from Arachis hypogaea alleviates salinity and drought stress in tobacco[J]. Plant Cell Reports,2015, 34(7):1109-1126.
[17] Ye N, Zhu G, Liu Y, et al. ABA controls H2O2 accumulation through the induction of OsCATB in rice leaves under water stress[J]. Plant&Cell Physiology,2011,52(4):689-698.
[18] Igarashi Y, Yoshiba Y, Sanada Y, et al. Characterization of the gene for deltal-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L[J].Plant Molecular Biology, 1997, 33(5):857-865.
[19] Hien D T, Jacobs M, Angenon G, et al. Proline accumulation and Alpyrroline-5-carboxylate synthetase gene properties in three rice cultivars differing in salinity and drought tolerance[J]. Plant Science,2003, 165(5):1059-1068.
[20] Sripinyowanich S, Klomsakul P, Boonburapong B, et al. Exogenous ABA induces salt tolerance in indica rice(Oryza sativa L.):The role of OsP5CS1 and OsP5CR gene expression during salt stress[J].Environmental&Experimental Botany, 2013, 86(2):94-105.
[21] Xiao B Z, Huang Y M, Tang N, et al. Over-expression of a LEA gene in rice improves drought resistance under the field conditions[J].Theoretical and Applied Genetics, 2007, 115(1):35-46.
[2] Odom A R, Stahlberg A, Wente S R, et al. A role for nuclear inositol1, 4, 5-trisphosphate kinase in transcriptional control[J]. Science,2000, 287(5460):2026-2029.
[3] Nalaskowski M M, Deschermeier C, Fanick W, et al. The human homologue of yeast ArgRIII protein is an inositol phosphate multikinase with predominantly nuclear localization[J]. Biochemical Journal, 2002, 366(2):549-556.
[4] Frederick J P, Mattiske D, Wofford J A, et al. An essential role for an inositol polyphosphate multikinase, Ipk2, in mouse embryogenesis and second messenger production[J]. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(24):8454-8459.
[5] Stevensonpaulik J, Odom A R, York J D. Molecular and biochemical characterization of two plant inositol polyphosphate 6-/3-/5-kinases[J]. Journal of Biological Chemistry, 2002, 277(45):42711-42718.
[6] Xia H J, Brearley C, Elge S, et al. Arabidopsis inositol polyphosphate6-/3-kinase is a nuclear protein that complements a yeast mutant lacking a functional argr-mcml transcription complex[J]. Plant Cell,2003, 15(2):449-463.
[7] Suzuki M, Tanaka K, Kuwano M, et al. Expression pattern of inositol phosphate-related enzymes in rice(Oryza sativa L.):implications for the phytic acid biosynthetic pathway[J]. Gene, 2007, 405(1-2):55-64.
[8] Caddick S E K, Harrison C J, Stavridou I, et al. A Solanumtuberosum inositol phosphate kinase(StITPK1)displaying inositol phosphate-inositol phosphate and inositol phosphate-ADP phosphotransferase activities[J]. Febs Letters, 2008, 582(12):1731-1737.
[9] Zhu J Q, Zhang J T, Tang R J, et al. Molecular characterization of ThIPK2, an inositol polyphosphate kinase gene homolog from Thellungiella halophila, and its heterologous expression to improve abiotic stress tolerance in Brassica napus[J]. Physiologia Plantarum,2009, 136(4):407-425.
[10] Yang L, Tang R, Zhu J, et al. Enhancement of stress tolerance in transgenic tobacco plants constitutively expressing AtIpk2β, an inositol polyphosphate 6-/3-kinase from Arabidopsis thaliana[J].Plant Molecular Biology,2008, 66(4):329-343.
[11] Liu M, Li D, Wang Z, et al. Transgenic expression of ThIPK2 gene in soybean improves stress tolerance, oleic acid content and seed size[J].Plant Cell Tissue&Organ Culture, 2012, 111(3):277-289.
[12] Zhu J K. Plant salt tolerance[J]. Trends in Plant Science, 2001, 6(2):66-71.
[13] Sairam R K, Srivastava G C, Agarwal S, et al. Differences in antioxidant activity in response to salinity stress in tolerant and susceptible wheat genotypes[J]. Biologia Plantarum, 2005, 49(1):85-91.
[14] Singh M, Kumar J, Singh S, et al. Roles of osmoprotectants in improving salinity and drought tolerance in plants:a review[J].Reviews in Environmental Science&Bio-technology, 2015, 14(3):407-426.
[15] Mittler R. Oxidative stress, antioxidants and stress tolerance[J].Trends in Plant Science, 2002, 7(9):405-410.
[16] Negi N P. Overexpression of CuZnSOD from Arachis hypogaea alleviates salinity and drought stress in tobacco[J]. Plant Cell Reports,2015, 34(7):1109-1126.
[17] Ye N, Zhu G, Liu Y, et al. ABA controls H2O2 accumulation through the induction of OsCATB in rice leaves under water stress[J]. Plant&Cell Physiology,2011,52(4):689-698.
[18] Igarashi Y, Yoshiba Y, Sanada Y, et al. Characterization of the gene for deltal-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L[J].Plant Molecular Biology, 1997, 33(5):857-865.
[19] Hien D T, Jacobs M, Angenon G, et al. Proline accumulation and Alpyrroline-5-carboxylate synthetase gene properties in three rice cultivars differing in salinity and drought tolerance[J]. Plant Science,2003, 165(5):1059-1068.
[20] Sripinyowanich S, Klomsakul P, Boonburapong B, et al. Exogenous ABA induces salt tolerance in indica rice(Oryza sativa L.):The role of OsP5CS1 and OsP5CR gene expression during salt stress[J].Environmental&Experimental Botany, 2013, 86(2):94-105.
[21] Xiao B Z, Huang Y M, Tang N, et al. Over-expression of a LEA gene in rice improves drought resistance under the field conditions[J].Theoretical and Applied Genetics, 2007, 115(1):35-46.