紫花苜蓿F-box蛋白基因MsFTL的克隆及功能分析
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摘要
FTL(F-box Triple LRR protein)是F-box蛋白家族的成员,具有F-box保守结构域,在植物抵御逆境胁迫过程中起重要作用。本研究参考低温胁迫下紫花苜蓿转录组数据设计引物,通过RT-PCR克隆获得紫花苜蓿MsFTL基因,该基因的全长1422 bp,编码473个氨基酸。该蛋白含有1个F-box结构域及3个LRR重复。系统进化分析表明,MsFTL与蒺藜苜蓿XP_003626345.1 F-box/FBD/LRR-repeat protein亲缘关系最近。两者蛋白序列比对发现共有11个差异位点。在低温、盐、干旱以及外源ABA处理下,MsFTL基因受到诱导,表达量上调。构建植物过表达载体pCBM-MsFTL,通过农杆菌介导法转化烟草。对经过抗性筛选、PCR和Real-time PCR验证的转基因植株进行低温抗性鉴定。在-4℃低温胁迫下,野生型烟草叶片出现了明显的萎蔫失水现象,而转基因烟草萎蔫程度相对较轻。生理检测结果表明,4℃处理24 h之后,转基因烟草的可溶性蛋白含量、可溶性糖含量、SOD活性,CAT活性高于野生型,MDA含量低于野生型。本研究表明,MsFTL基因在提高植物对低温胁迫的抗性方面具有重要的作用。
FTL(F-box Triple LRR)protein belongs to the family of F-box proteins that play an important role in tolerance to low temperature. In this study,an MsFTL gene was isolated by candidate gene approach from alfalfa,and this gene was differently expressed in leaf under cold stress. The full-length cDNA of MsFTL gene was 1422 bp,which putatively encoded for 473 amino acids. By bioinformatic analysis,the MsFTL protein was found to carry an F-box domain and three LRR repeats in C-terminus. MsFTL showed homology closely to XP_003626345.1,a member of F-box/FBD/LRR-repeat protein in Medicago truncatula,with 11 differential sites between the two proteins. The expression of MsFTL was induced by low temperature,salt,drought stress and ABA treatments. We generated the transformation construct and transformed the MsFTL into tobacco. Under-4 ℃,wild-type tobacco leaves showed remarkable wilting,while the transgenic plants presented slightly stressed phenotype. Transgenic plants accumulated higher content of soluble protein and soluble sugar than that of wildtype. The activity of SOD and CAT in transgenic plants was higher than those of wild type under low temperature(4 ℃)for 24 h. Moreover,the MDA content in transgenic plants was lower than that of wild type. Thus,these results suggested that overexpressing MsFTL might significantly improve tobacco plant tolerance to low temperature stress.
FTL(F-box Triple LRR)protein belongs to the family of F-box proteins that play an important role in tolerance to low temperature. In this study,an MsFTL gene was isolated by candidate gene approach from alfalfa,and this gene was differently expressed in leaf under cold stress. The full-length cDNA of MsFTL gene was 1422 bp,which putatively encoded for 473 amino acids. By bioinformatic analysis,the MsFTL protein was found to carry an F-box domain and three LRR repeats in C-terminus. MsFTL showed homology closely to XP_003626345.1,a member of F-box/FBD/LRR-repeat protein in Medicago truncatula,with 11 differential sites between the two proteins. The expression of MsFTL was induced by low temperature,salt,drought stress and ABA treatments. We generated the transformation construct and transformed the MsFTL into tobacco. Under-4 ℃,wild-type tobacco leaves showed remarkable wilting,while the transgenic plants presented slightly stressed phenotype. Transgenic plants accumulated higher content of soluble protein and soluble sugar than that of wildtype. The activity of SOD and CAT in transgenic plants was higher than those of wild type under low temperature(4 ℃)for 24 h. Moreover,the MDA content in transgenic plants was lower than that of wild type. Thus,these results suggested that overexpressing MsFTL might significantly improve tobacco plant tolerance to low temperature stress.
引文
[1]许克恒,张云彤,张莹,王彬,王法微,李海燕·植物F-box基因家族的研究进展·生物技术通报,2018, 32(1):26-32Xu K H, Zhang Y T, Zhang Y, Wang B, Wang F W, Li H Y. Research progress of plant F-box gene family. Biotechnol Bulletin, 2018,32(1):26-32
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[3]Jin J,Cardozo T,Lovering R C,Elledge S J,Pagano M,Harper J W. Systematic analysis and nomenclature of mammalian F-box proteins. Genes&Development, 2004, 18(21):2573-2580
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[5]霍冬英,郑炜君,李盼松,徐兆师,周永斌,陈明,马有志,闵东红,张小红·谷子F-Box家族基因的鉴定,分类及干旱响应·作物学报,2014,40(9):1585-1594Huo D Y, Zheng W J, Li P S, Xu Z S, Zhou Y B, Chen M,Ma Y Z, Min D H, Zhang X H. Millet F-Box family gene identification, classification and drought response-Acta Agronomica Sinica, 2014, 40(9):1585-1594
[6]Jain M, Ni A, Arora R, Agarwal P, Ray S, Harma P S, Kapoor S,Tyagi A K, Hurana J P K. F-Box proteins in rice. Genome-wide analysis, classification, temporal and spatial gene expression during panicle and seed development, and regulation by light and abiotic stress. Plant Physiology, 2007, 143(4):1467-1483
[7]Tyler L,Thomas S G,Hu J,Dill A,Alonso J M,Ecker J R, Sun T-DELLA proteins and gibberellin-regulated seed germination and floral development in Arabidopsis. PlantPhysiology, 2004, 135(38):1008-1019
[8] Xu G Y,Cui Y C,Wang M L. OsMsr9, a novel putative rice F-box containing protein, confers enhanced salt tolerance in transgenic rice and Arabidopsis. Molecular Breeding, 2014,34(21):1055-1064
[9] Min J H, Dae Y K, Si Y K, Dong S K, Jin B K, Yong W S. Wheat F-box protein recruits proteins and regulates their abundance during wheat spike development. Molecular Biology Reports, 2012,42(18):132-143
[10] Chen Z H, Li M T, Yuan Y. Ectopic expression of cucumber(Cucumis sativus L.)CsTIR/AFB genes enhance salt tolerance in transgenic Arabidopsis. Plant Cell Tissue and Organ Culture,2017, 131(1):107-118
[11] Chen R, Guo W, Yin Y, Gong Z H. A novel F-Box protein CaF-Box is involved in responses to plant hormones and abiotic stress in Pepper(Capsicum annuum L.)·International Journal of Molecular Sciences, 2014, 15(2):2413-2430
[12]曹宏,章会玲,盖琼辉,陈红,赵满来. 22个紫花苜蓿品种的引种试验和生产性能综合评价·草业学报,2011,20(6):219-229Cao H, Zhang H L,Gai Q H,Chen H,Zhao M L.Comprehensive evaluation of introduction and performance of22 alfalfa varieties. Journal of Grassland Science, 2011,20(6):219-229
[13]隋勇,刘长恩·肇东市苜蓿产业化发展的现状与对策·中国乳业,2012,21(5):11-12Sui Y, Liu C E. Current situation and countermeasures of Alfalfa industrialization in Zhaodong city. China Dairy Industry, 2012,21(5):11-12
[14] Lu G Q, Moriyama E N-Vector NTI, a balanced all-in-one sequence analysis suite. Briefings in Bioinformatics, 2004, 5(4):378-388
[15] Thompson J D, Gibson T J, Plewniak F. The CLUSTAL_X windows interface:flexible strategies for multiple sequence align-ment aided by quality analysis tools. Nucleic Acids Research, 1997,25(24):4876-4882
[16] Tamura K, Peterson D, Peterson N. MEGA5:Molecular evo-lutionary genetics analysis using maximum likelihood,evolutionarydistance, and maximum parsimony methods.Molecular Biology and Evolution, 2011,28(10):2731-2739
[17] Hoekema A, Hirsch P R, Hooykaas P J. A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature, 1983,303(5913):179-180
[18] Zhao L N,Liu F X,Xu W Y,Di C,Zhou S X,Xue Y B,Yu J J, Su Z. Increased expression of OsSPX1 enhances cold/subfreezing tolerance in tobacco and Arabidopsis thaliana. Plant Biotechnology Journal, 2009, 7(7):550-561
[19] Dhindsa R S, Matowe W. Drought tolerance in two mosses:correlated with enzymatic defence against lipid peroxidationJournal of Experimental Botany, 1981,32(1):79-91
[20] Shi J,Fu X Z,Peng T,Huang X S,Fan Q J,Liu J H. Spermine pretreatment confers dehydration tolerance of citrus in vitro plants via modulation of antioxidative capacity and stomatal response. Tree Physiology,2010, 30(7):914-922
[21] Aebi H. Catalase in vitro. Methods in Enzymology, 1984, 105(105):121-126
[22] Bai C, Sen P, Hofmann K, Ma L, Goebl M, Harper J W,Elledge S J. SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell,1996, 86(2):263-274
[23] Edward T K, Michele P. The F-box protein family. Genome Biology,2000, 1(5):1-7
[24]于兰芳,周硕,张双喜,纪玉洁,刘永伟·葡萄F-box基因VvF-box5的基因结构与表达分析·植物遗传资源学报,2018,19(2):361-369Yu L F, Zhou S, Zhang S X, Ji Y J, Liu Y W. Analysis of gene structure and expression of f-box gene VvF-box5 in grapeJournal of Plant Genetic Resources, 2018, 19(2):361-369
[25] Cui H R, Zhang Z R, Lv W. Genome-wide characterization and analysis of F-box protein-encoding genes in the Malus domestica genome. Molecular Genetics and Genomics, 2015,290(4):1435-1446
[26] Jia F,Wu B,Li H. Genome-wide identification and characterisation of F-box family in maize. Molecular Genetics and Genomics, 2013,288(11):559-577
[27] Li Y, Jia F, Yu Y. The SCF E3 ligase AtPP2-B11 plays a negative role in response to drought stress in Arabidopsis-Plant Molecular Biology Reporter, 2014, 32(5):943-956
[28] Zhao Z X, Zhang G Q, Zhou S M. The improvement of salt tolerance in transgenic tobacco by overexpression of wheat F-box gene TaFBA1. Plant Science, 2017(259):71-85
[29] An J P, Li R, Qu F J-Apple F-Box protein MdMAX2 regulates plant photomorphogenesis and stress response. Frontiers in Plant Science, 2016, 7(1235):1685-1699
[30] Hou L X, Zhang G K, Zhao F G. VvBAPl is involved in cold tolerance in Vitis vinifera L. Frontiers in Plant Science, 2018,9(6):102-123
[31] Gu L J, Cheng H M. Isolation, molecular cloning and characterization of a cold-responsive gene, AmDUF1517, from Ammopiptanthus mongolicus. Plant Cell, Tissue and Organ Culture,2014, 117(2):201-211
[32]Mccord J M,fridovich I. Speroxide dismutase:an enzymic function for erythrocuprein(hemocuprein). Journal of Biological Chemistry, 1969,224(22):6049-6055
[33] Shalata A,Neumann P M. Exogenous ascorbic acid(vitamin C)increases resistance to salt stress and reduces lipid peroxidation.Journal of Experimental Botany, 2001,52(364):2207-2211
[34] Zhu X, Li Q, Hu J, Wang M, Li X. Molecular cloning and characterization of spermine synthesis gene associated with cold tolerance in Tea plant(Camellia sinensis)·Applied Biochemistry and Biotechnology, 2015, 177(5):1055-1068
[35] Steponkus P L. Role of the plasma membrane in freezing injury and cold acclimation. Annual Review of Plant Biology, 2003,35(1):543-584
[36]Sagarika M,Sanjeev K,Bedabrata S. Crosstalk between salt,drought, and cold stress in plants:Toward genetic engineering for stress tolerance. Abiotic Stress Resports Plant,2016,23(5):57-88
[37]Nelofer J,Umer M,Khurshid I,Riffat J. Cold stress modulates osmolytes and antioxidant system in Calendula officinalis. Acta Physiologiae Plantarum, 2018,40(4):73-86
[2]Koo A J, Thireault C, Zemelis S, Poudel A N, Zhang T,Kitaoka N F, Brandizzi M H, Howe G A-Endoplasmic reticulum-associated inactivation of the hormone jasmonoylL-isoleucine by multiple members of the cytochrome P45094family in Arabidopsis-Journal of Biological Chemistry, 2014,289(43):29728-29738
[3]Jin J,Cardozo T,Lovering R C,Elledge S J,Pagano M,Harper J W. Systematic analysis and nomenclature of mammalian F-box proteins. Genes&Development, 2004, 18(21):2573-2580
[4]Jana J R,Vignesh D,Parameswari P,Sangeeth P D,Su R C,So Y Y, Seongmin H, Sang H O, Man H O, Yong P L. F-Box genes in Brassica rapa:genome-wide identification, structural characterization, expressional validation, and comparative analysis. Plant Molecular Biology Reporter, 2018,36(3):500-517
[5]霍冬英,郑炜君,李盼松,徐兆师,周永斌,陈明,马有志,闵东红,张小红·谷子F-Box家族基因的鉴定,分类及干旱响应·作物学报,2014,40(9):1585-1594Huo D Y, Zheng W J, Li P S, Xu Z S, Zhou Y B, Chen M,Ma Y Z, Min D H, Zhang X H. Millet F-Box family gene identification, classification and drought response-Acta Agronomica Sinica, 2014, 40(9):1585-1594
[6]Jain M, Ni A, Arora R, Agarwal P, Ray S, Harma P S, Kapoor S,Tyagi A K, Hurana J P K. F-Box proteins in rice. Genome-wide analysis, classification, temporal and spatial gene expression during panicle and seed development, and regulation by light and abiotic stress. Plant Physiology, 2007, 143(4):1467-1483
[7]Tyler L,Thomas S G,Hu J,Dill A,Alonso J M,Ecker J R, Sun T-DELLA proteins and gibberellin-regulated seed germination and floral development in Arabidopsis. PlantPhysiology, 2004, 135(38):1008-1019
[8] Xu G Y,Cui Y C,Wang M L. OsMsr9, a novel putative rice F-box containing protein, confers enhanced salt tolerance in transgenic rice and Arabidopsis. Molecular Breeding, 2014,34(21):1055-1064
[9] Min J H, Dae Y K, Si Y K, Dong S K, Jin B K, Yong W S. Wheat F-box protein recruits proteins and regulates their abundance during wheat spike development. Molecular Biology Reports, 2012,42(18):132-143
[10] Chen Z H, Li M T, Yuan Y. Ectopic expression of cucumber(Cucumis sativus L.)CsTIR/AFB genes enhance salt tolerance in transgenic Arabidopsis. Plant Cell Tissue and Organ Culture,2017, 131(1):107-118
[11] Chen R, Guo W, Yin Y, Gong Z H. A novel F-Box protein CaF-Box is involved in responses to plant hormones and abiotic stress in Pepper(Capsicum annuum L.)·International Journal of Molecular Sciences, 2014, 15(2):2413-2430
[12]曹宏,章会玲,盖琼辉,陈红,赵满来. 22个紫花苜蓿品种的引种试验和生产性能综合评价·草业学报,2011,20(6):219-229Cao H, Zhang H L,Gai Q H,Chen H,Zhao M L.Comprehensive evaluation of introduction and performance of22 alfalfa varieties. Journal of Grassland Science, 2011,20(6):219-229
[13]隋勇,刘长恩·肇东市苜蓿产业化发展的现状与对策·中国乳业,2012,21(5):11-12Sui Y, Liu C E. Current situation and countermeasures of Alfalfa industrialization in Zhaodong city. China Dairy Industry, 2012,21(5):11-12
[14] Lu G Q, Moriyama E N-Vector NTI, a balanced all-in-one sequence analysis suite. Briefings in Bioinformatics, 2004, 5(4):378-388
[15] Thompson J D, Gibson T J, Plewniak F. The CLUSTAL_X windows interface:flexible strategies for multiple sequence align-ment aided by quality analysis tools. Nucleic Acids Research, 1997,25(24):4876-4882
[16] Tamura K, Peterson D, Peterson N. MEGA5:Molecular evo-lutionary genetics analysis using maximum likelihood,evolutionarydistance, and maximum parsimony methods.Molecular Biology and Evolution, 2011,28(10):2731-2739
[17] Hoekema A, Hirsch P R, Hooykaas P J. A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature, 1983,303(5913):179-180
[18] Zhao L N,Liu F X,Xu W Y,Di C,Zhou S X,Xue Y B,Yu J J, Su Z. Increased expression of OsSPX1 enhances cold/subfreezing tolerance in tobacco and Arabidopsis thaliana. Plant Biotechnology Journal, 2009, 7(7):550-561
[19] Dhindsa R S, Matowe W. Drought tolerance in two mosses:correlated with enzymatic defence against lipid peroxidationJournal of Experimental Botany, 1981,32(1):79-91
[20] Shi J,Fu X Z,Peng T,Huang X S,Fan Q J,Liu J H. Spermine pretreatment confers dehydration tolerance of citrus in vitro plants via modulation of antioxidative capacity and stomatal response. Tree Physiology,2010, 30(7):914-922
[21] Aebi H. Catalase in vitro. Methods in Enzymology, 1984, 105(105):121-126
[22] Bai C, Sen P, Hofmann K, Ma L, Goebl M, Harper J W,Elledge S J. SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell,1996, 86(2):263-274
[23] Edward T K, Michele P. The F-box protein family. Genome Biology,2000, 1(5):1-7
[24]于兰芳,周硕,张双喜,纪玉洁,刘永伟·葡萄F-box基因VvF-box5的基因结构与表达分析·植物遗传资源学报,2018,19(2):361-369Yu L F, Zhou S, Zhang S X, Ji Y J, Liu Y W. Analysis of gene structure and expression of f-box gene VvF-box5 in grapeJournal of Plant Genetic Resources, 2018, 19(2):361-369
[25] Cui H R, Zhang Z R, Lv W. Genome-wide characterization and analysis of F-box protein-encoding genes in the Malus domestica genome. Molecular Genetics and Genomics, 2015,290(4):1435-1446
[26] Jia F,Wu B,Li H. Genome-wide identification and characterisation of F-box family in maize. Molecular Genetics and Genomics, 2013,288(11):559-577
[27] Li Y, Jia F, Yu Y. The SCF E3 ligase AtPP2-B11 plays a negative role in response to drought stress in Arabidopsis-Plant Molecular Biology Reporter, 2014, 32(5):943-956
[28] Zhao Z X, Zhang G Q, Zhou S M. The improvement of salt tolerance in transgenic tobacco by overexpression of wheat F-box gene TaFBA1. Plant Science, 2017(259):71-85
[29] An J P, Li R, Qu F J-Apple F-Box protein MdMAX2 regulates plant photomorphogenesis and stress response. Frontiers in Plant Science, 2016, 7(1235):1685-1699
[30] Hou L X, Zhang G K, Zhao F G. VvBAPl is involved in cold tolerance in Vitis vinifera L. Frontiers in Plant Science, 2018,9(6):102-123
[31] Gu L J, Cheng H M. Isolation, molecular cloning and characterization of a cold-responsive gene, AmDUF1517, from Ammopiptanthus mongolicus. Plant Cell, Tissue and Organ Culture,2014, 117(2):201-211
[32]Mccord J M,fridovich I. Speroxide dismutase:an enzymic function for erythrocuprein(hemocuprein). Journal of Biological Chemistry, 1969,224(22):6049-6055
[33] Shalata A,Neumann P M. Exogenous ascorbic acid(vitamin C)increases resistance to salt stress and reduces lipid peroxidation.Journal of Experimental Botany, 2001,52(364):2207-2211
[34] Zhu X, Li Q, Hu J, Wang M, Li X. Molecular cloning and characterization of spermine synthesis gene associated with cold tolerance in Tea plant(Camellia sinensis)·Applied Biochemistry and Biotechnology, 2015, 177(5):1055-1068
[35] Steponkus P L. Role of the plasma membrane in freezing injury and cold acclimation. Annual Review of Plant Biology, 2003,35(1):543-584
[36]Sagarika M,Sanjeev K,Bedabrata S. Crosstalk between salt,drought, and cold stress in plants:Toward genetic engineering for stress tolerance. Abiotic Stress Resports Plant,2016,23(5):57-88
[37]Nelofer J,Umer M,Khurshid I,Riffat J. Cold stress modulates osmolytes and antioxidant system in Calendula officinalis. Acta Physiologiae Plantarum, 2018,40(4):73-86