转紫花苜蓿MsLEA2基因提高拟南芥耐铝毒性研究
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摘要
MsLEA2基因是从紫花苜蓿中克隆到的胚胎晚期富集蛋白基因,属于LEA_2家族。以转基因拟南芥T_3代植株的3个株系为材料,从表型、生理和分子生物学三个方面研究铝胁迫下转MsLEA2基因拟南芥的耐铝毒性能。结果表明:铝胁迫下转基因株系的脯氨酸含量高于对照(野生型),丙二醛含量和电导率则低于对照且差异显著(P<0.05),CAT、POD和SOD活性显著高于对照。初步证明转MsLEA2基因拟南芥的耐铝毒性能明显高于对照,紫花苜蓿的MsLEA2基因具有提高植物耐铝毒胁迫的能力。
The MsLEA2 gene is an late embryogenesis abundant(LEA) protein gene cloned from alfalfa and belongs to the LEA_2 family. In order to identify the resistant aluminum toxicity of the MsLEA2 gene, three strains of transgenic Arabidopsis T_3 plants were used as materials to study the resistance of transgenic Arabidopsis thaliana under aluminum stress from phenotype, physiology and molecular biology. The results showed that the proline content of the transgenic lines was higher than that of the control(wild-type) under the aluminum stress, while the content of malondialdehyde and conductivity was lower than that of the control plants, and the difference was significant(P<0.05); the CAT, POD and SOD enzyme activities of the transgenic lines were significantly higher than the control. These results preliminarily proved that the aluminum tolerance of MsLEA2 transgenic Arabidopsis thaliana was significantly higher than that of the control. The MsLEA2 gene of alfalfa had the ability to increase the tolerance to aluminum toxicity of plants.
The MsLEA2 gene is an late embryogenesis abundant(LEA) protein gene cloned from alfalfa and belongs to the LEA_2 family. In order to identify the resistant aluminum toxicity of the MsLEA2 gene, three strains of transgenic Arabidopsis T_3 plants were used as materials to study the resistance of transgenic Arabidopsis thaliana under aluminum stress from phenotype, physiology and molecular biology. The results showed that the proline content of the transgenic lines was higher than that of the control(wild-type) under the aluminum stress, while the content of malondialdehyde and conductivity was lower than that of the control plants, and the difference was significant(P<0.05); the CAT, POD and SOD enzyme activities of the transgenic lines were significantly higher than the control. These results preliminarily proved that the aluminum tolerance of MsLEA2 transgenic Arabidopsis thaliana was significantly higher than that of the control. The MsLEA2 gene of alfalfa had the ability to increase the tolerance to aluminum toxicity of plants.
引文
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[3] Mowla S, Cuypers A, Driscoll S, et al. Yeast complementation reveals a role for an Arabidopsis thaliana late embryogenesis abundant(LEA)-like protein in oxidative stress tolerance[J]. Plant Journal, 2010, 48(5):743-756.
[4] 何道一,周秀杰,张坤亮,等.大豆Lea5基因的克隆与表达分析[J].激光生物学报, 2017(6):550-556.He Daoyi, Zhou Xiujie, Zhang Kunliang, et al. Cloning and expression analysis of soybean Lea5 gene[J]. Chinese Journal of Laser Biology, 2017(6): 550-556.
[5] Zhang J, Duan Z, Zhang D, et al. Co-transforming bar and CsLEA enhanced tolerance to drought and salt stress in transgenic alfalfa(Medicago sativa L.)[J]. Biochemical & Biophysical Research Communications, 2016, 472(1):75-82.
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[10] 张志飞,龚梨霞,文昭竹,等.酸铜对紫花苜蓿种子萌发及根系生长的影响[J].中国草地学报, 2017, 39(3):72-76.Zhang Zhifei, Gong Lixia, Wen Zhaozhu, et al.Effect of acid copper on seed germination and root growth of alfalfa[J]. Chinese Journal of Grassland, 2014, 36(5): 38-45.
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[12] Hunault G, Jaspard E. LEAPdb: a database for the late embryogenesis abundant proteins [J]. BMC Genomics, 2010, 11(1):221-0.
[13] Zhang Y. I-TASSER: Fully automated protein structure prediction in CASP8[J]. Proteins Structure Function & Bioinformatics, 2010, 77(S9):100-113.
[14] Bies-Ethève N, Gaubier-Comella P, Debures A, et al. Inventory, evolution and expression profiling diversity of the LEA(late embryogenesis abundant) protein gene family in Arabidopsis thaliana[J]. Plant Molecular Biology, 2008, 67(1-2):107-124.
[15] Vinarov D A, Loushin C N, Markley J L. Wheat germ cell-free platform for eukaryotic protein production[J]. Febs Journal, 2010, 273(18):4160-4169.
[16] Singh S, Cornilescu C C, Tyler R C, et al. Solution structure of a late embryogenesis abundant protein (LEA14) from Arabidopsis thaliana, a cellular stress-related protein[J]. Protein Science, 2010, 14(10):2601-2609.
[17] Ghorbanpour A, Salimi A, Ali M, et al.The effect of Trichoderma harzianum in mitigating low temperature stress in tomato(Solanum lycopersicum L.)plants[J].Scientia Horticulturae, 2018, 230:134-141.
[18] Shobbar Z S, Oane R, Gamuyao R, et al. Abscisic acid regulates gene expression in cortical fiber cells and silica cells of rice shoots[J]. New Phytologist, 2010, 178(1):68-79.
[19] Ma B, Gao L, Zhang H, et al. Aluminum-induced oxidative stress and changes in antioxidant defenses in the roots of rice varieties differing in Al tolerance[J]. Plant Cell Reports, 2012, 31(4):687-696.
[20] Liu H, Yu C, Li H, et al. Overexpression of ShDHN, a dehydrin gene from Solanum habrochaites enhances tolerance to multiple abiotic stresses in tomato[J]. Plant Science, 2015, 231:198-211.
[21] Zhang X, Lu S, Jiang C, et al. RcLEA, a late embryogenesis abundant protein gene isolated from Rosa chinensis, confers tolerance to Escherichia coli and Arabidopsis thaliana and stabilizes enzyme activity under diverse stresses[J]. Plant Molecular Biology, 2014, 85(4-5):333-347.
[2] Saha B, Mishra S, Awasthi J P, et al. Enhanced drought and salinity tolerance in transgenic mustard[Brassica juncea(L.)Czern&Coss.] overexpressing Arabidopsis, group 4 late embryogenesis abundant gene(AtLEA4-1)[J]. Environmental & Experimental Botany, 2016, 128:99-111.
[3] Mowla S, Cuypers A, Driscoll S, et al. Yeast complementation reveals a role for an Arabidopsis thaliana late embryogenesis abundant(LEA)-like protein in oxidative stress tolerance[J]. Plant Journal, 2010, 48(5):743-756.
[4] 何道一,周秀杰,张坤亮,等.大豆Lea5基因的克隆与表达分析[J].激光生物学报, 2017(6):550-556.He Daoyi, Zhou Xiujie, Zhang Kunliang, et al. Cloning and expression analysis of soybean Lea5 gene[J]. Chinese Journal of Laser Biology, 2017(6): 550-556.
[5] Zhang J, Duan Z, Zhang D, et al. Co-transforming bar and CsLEA enhanced tolerance to drought and salt stress in transgenic alfalfa(Medicago sativa L.)[J]. Biochemical & Biophysical Research Communications, 2016, 472(1):75-82.
[6] Dang N X, Popova A V, Hundertmark M, et al. Functional characterization of selected LEA proteins from Arabidopsis thaliana, in yeast and in vitro[J]. Planta, 2014, 240(2):325-36.
[7] Foy C D, And R L C, White M C. The physiology of metal toxicity in plants[J]. Annu. Rev. Plant Physiol., 1978, 29(1):511-566.
[8] 马力,周鹏,高小叶,等.非秋眠和半秋眠紫花苜蓿品种在华东冬闲田的生长规律和营养动态[J].中国草地学报, 2014, 36(5):38-45.Ma Li, Zhou Peng, Gao Xiaoye, et al. Growth and nutrition dynamics of non-fall dormancy and semi-fall dormancy alfalfa cultivars in winter fallow land of south-eastchina[J].Chinese Journal of Grassland, 2014, 36(5): 38-45.
[9] 肖玉,贾婷婷,李天银,等.交替沟灌对紫花苜蓿产量和品质的影响[J].中国草地学报,2015, 37(6):42-48.Xiao Yu, Jia Tingting, Li Tianyin, et al. Effects of alternate irrigation on alfalfa(Medicago sativa) yield and quality[J].Chinese Journal of Grassland, 2015, 37(6): 43-48.
[10] 张志飞,龚梨霞,文昭竹,等.酸铜对紫花苜蓿种子萌发及根系生长的影响[J].中国草地学报, 2017, 39(3):72-76.Zhang Zhifei, Gong Lixia, Wen Zhaozhu, et al.Effect of acid copper on seed germination and root growth of alfalfa[J]. Chinese Journal of Grassland, 2014, 36(5): 38-45.
[11] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method[J]. Methods, 2001, 25(4):402-408.
[12] Hunault G, Jaspard E. LEAPdb: a database for the late embryogenesis abundant proteins [J]. BMC Genomics, 2010, 11(1):221-0.
[13] Zhang Y. I-TASSER: Fully automated protein structure prediction in CASP8[J]. Proteins Structure Function & Bioinformatics, 2010, 77(S9):100-113.
[14] Bies-Ethève N, Gaubier-Comella P, Debures A, et al. Inventory, evolution and expression profiling diversity of the LEA(late embryogenesis abundant) protein gene family in Arabidopsis thaliana[J]. Plant Molecular Biology, 2008, 67(1-2):107-124.
[15] Vinarov D A, Loushin C N, Markley J L. Wheat germ cell-free platform for eukaryotic protein production[J]. Febs Journal, 2010, 273(18):4160-4169.
[16] Singh S, Cornilescu C C, Tyler R C, et al. Solution structure of a late embryogenesis abundant protein (LEA14) from Arabidopsis thaliana, a cellular stress-related protein[J]. Protein Science, 2010, 14(10):2601-2609.
[17] Ghorbanpour A, Salimi A, Ali M, et al.The effect of Trichoderma harzianum in mitigating low temperature stress in tomato(Solanum lycopersicum L.)plants[J].Scientia Horticulturae, 2018, 230:134-141.
[18] Shobbar Z S, Oane R, Gamuyao R, et al. Abscisic acid regulates gene expression in cortical fiber cells and silica cells of rice shoots[J]. New Phytologist, 2010, 178(1):68-79.
[19] Ma B, Gao L, Zhang H, et al. Aluminum-induced oxidative stress and changes in antioxidant defenses in the roots of rice varieties differing in Al tolerance[J]. Plant Cell Reports, 2012, 31(4):687-696.
[20] Liu H, Yu C, Li H, et al. Overexpression of ShDHN, a dehydrin gene from Solanum habrochaites enhances tolerance to multiple abiotic stresses in tomato[J]. Plant Science, 2015, 231:198-211.
[21] Zhang X, Lu S, Jiang C, et al. RcLEA, a late embryogenesis abundant protein gene isolated from Rosa chinensis, confers tolerance to Escherichia coli and Arabidopsis thaliana and stabilizes enzyme activity under diverse stresses[J]. Plant Molecular Biology, 2014, 85(4-5):333-347.
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