陆地棉GhLEA3基因的克隆及其响应低温胁迫表达分析
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摘要
【目的】本研究旨在探索棉花GhLEA3基因的结构特征及其在低温胁迫下的表达模式,研究其抗逆功能。【方法】利用同源序列克隆法在陆地棉中克隆GhLEA3;采用生物信息学方法分析其蛋白质性质,构建系统进化树。采用In-Fusion连接技术构建融合蛋白表达载体35S∷GhLEA3-GFP并进行亚细胞定位;在棉花三叶期进行叶片低温表达分析;采用农杆菌介导的花序浸染法将GhLEA3基因转入拟南芥;对T_3转基因拟南芥种子进行4℃低温萌发试验;低温处理后测转基因拟南芥叶片的电导率。【结果】GhLEA3基因编码序列全长1 218bp,编码405个氨基酸;GhLEA3蛋白含有4个功能结构域PF02987。陆地棉GhLEA3与拟南芥AtLEA3氨基酸序列之间相似性为52.6%。GhLEA3蛋白可能定位在液泡和小泡中。GhLEA3基因在低温处理的棉花叶片中上调表达。T_3转基因拟南芥低温萌发率显著高于野生型,低温处理后转基因拟南芥叶片电导率显著低于野生型。【结论】陆地棉GhLEA3属于典型的LEA3家族成员,与拟南芥AtLEA3亲缘关系最近;该基因响应低温胁迫,可能在抗冷中起重要作用。
[Objective] Based on the analysis of cotton GhLEA3 gene structure and its expression model in chilling stress, we investigated the resistance function of GhLEA3 in response to cold stress. [Method] We cloned GhLEA3 from upland cotton by homologous sequence cloning way. We analyzed the properties of the GhLEA3, and constructed phylogenetic tree by bioinformatics analysis. We constructed the transient expression vector 35 S∷GhLEA3-GFP by In-Fusion connection technology and studied the subcellular localization of GhLEA3. The expression of GhLEA3 gene in leaves of TM-1 at three-leaf stage under low temperature stress treatments(4 ℃, 24 h) was performed. Agrobacterium tumefaciens mediated floral dipping method was used to transform the gene with expression vector 35 S ∷ GhLEA3-GFP into Arabidopsis thaliana wild type. Low temperature germination experiment at 4 ℃ was conducted to verify germination ability of the transgenic Arabidopsis thaliana T_3 seed. The transgenic Arabidopsis seedlings were treated at 4 ℃(low temperature), and all the leaves were taken to measure their electrical conductivity. [Result] The coding sequence of GhLEA3 gene is 1 218 bp, which encodes 405 amino acids. GhLEA3 protein includes 4 functional domains of PF02987. Phylogenetic analysis showed that the similarity of amino acid sequences between upland cotton GhLEA3 and Arabidopsis thaliana AtLEA3 was 52.6%. GhLEA3 might be mainly localized in vacuoles and small vesicles. GhLEA3 was up-regulated in leaves after low temperature treatment. The germination rate of transgenic Arabidopsis thaliana of T_3 generation at low temperature was significantly higher than that of wild type, and its electrical conductivity of leaves after low temperature treatment was significantly lower than that of wild type. [Conclusion] GhLEA3 belonged to the member of LEA3 family. Phylogenetic analysis showed that GhLEA3 had the closest relationship with AtLEA3. GhLEA3 was induced by low temperature stress and may play an important role in improving cold resistance.
[Objective] Based on the analysis of cotton GhLEA3 gene structure and its expression model in chilling stress, we investigated the resistance function of GhLEA3 in response to cold stress. [Method] We cloned GhLEA3 from upland cotton by homologous sequence cloning way. We analyzed the properties of the GhLEA3, and constructed phylogenetic tree by bioinformatics analysis. We constructed the transient expression vector 35 S∷GhLEA3-GFP by In-Fusion connection technology and studied the subcellular localization of GhLEA3. The expression of GhLEA3 gene in leaves of TM-1 at three-leaf stage under low temperature stress treatments(4 ℃, 24 h) was performed. Agrobacterium tumefaciens mediated floral dipping method was used to transform the gene with expression vector 35 S ∷ GhLEA3-GFP into Arabidopsis thaliana wild type. Low temperature germination experiment at 4 ℃ was conducted to verify germination ability of the transgenic Arabidopsis thaliana T_3 seed. The transgenic Arabidopsis seedlings were treated at 4 ℃(low temperature), and all the leaves were taken to measure their electrical conductivity. [Result] The coding sequence of GhLEA3 gene is 1 218 bp, which encodes 405 amino acids. GhLEA3 protein includes 4 functional domains of PF02987. Phylogenetic analysis showed that the similarity of amino acid sequences between upland cotton GhLEA3 and Arabidopsis thaliana AtLEA3 was 52.6%. GhLEA3 might be mainly localized in vacuoles and small vesicles. GhLEA3 was up-regulated in leaves after low temperature treatment. The germination rate of transgenic Arabidopsis thaliana of T_3 generation at low temperature was significantly higher than that of wild type, and its electrical conductivity of leaves after low temperature treatment was significantly lower than that of wild type. [Conclusion] GhLEA3 belonged to the member of LEA3 family. Phylogenetic analysis showed that GhLEA3 had the closest relationship with AtLEA3. GhLEA3 was induced by low temperature stress and may play an important role in improving cold resistance.
引文
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[32]Liu Yang,Liang Jianan,Sun Liping,et al.Group 3 LEA protein,ZmLEA3,is involved in protection from low temperature stress[J/OL].Frontiers in Plant Science,2016,7:1011[2018-01-01].https://doi.org/10.3389/fpls.2016.01011
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[36]Hsing Y C,Chen Z Y,Shih M D,et al.Unusual sequences of group 3 LEA mRNA inducible by maturation or drying in soybean seeds[J/OL].Plant Molecular Biology,1995,29(4):863-868[2018-01-01].https://doi.org/10.1007/BF00041175
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[2]Thomashow M F.Plant cold acclimation:Freezing tolerance genes and regulatory mechanisms[J].Annual Review of Plant Biology,2003,50(50):571-599.
[3]Singh V,Singh A P,Bhadoria J,et al.Differential expression of salt-responsive genes to salinity stress in salt-tolerant and salt-sensitive rice(Oryza sativa L.)at seedling stage[J].Protoplasma,2018,255(6):1667-1681.
[4]Hundertmark M,Hincha D K.LEA(late embryogenesis abundant)proteins and their encoding genes in Arabidopsis thaliana[J/OL].BMC Genomics,2008,9(1):118(2008-03-04)[2018-01-01].http://dx.doi.org/10.1186/1471-2164-9-118
[5]Dure L,Greenway S C,Galau G A.Developmental biochemistry of cottonseed embryogenesis and germination:Changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein synthesis[J/OL].Biochemistry,1981,20(14):4162-4168[2018-01-01].https://doi.org/10.1021/bi00517a033
[6]Galau G A.Developmental biochemistry of cottonseed embryogenesis and germination:Changing messenger ribonucleic acid populations as shown by reciprocal heterologous complementary deoxyribonucleic acid-messenger ribonucleic acid hybridization[J/OL].Biochemistry,1981,20(14):4169-4178[2018-01-01].https://doi.org/10.1021/bi00517a033
[7]Galau G A,Hughes D W,Dure L III.Abscisic acid induction of cloned cotton late embryogenesis-abundant(LEA)mRNAs[J/OL].Plant Molecular Biology,1986,7(3):155-170[2018-01-01].https://doi.org/10.1007/BF00021327
[8]Dure L,Crouch M,Harada J,et al.Common amino acid sequence domains among the LEA proteins of higher plants[J/OL].Plant Molecular Biology,1989,12(5):475-486[2018-01-01].https://doi.org/10.1007/bf00036962
[9]Xue Rong,Liu Yun,Zheng Yizhi,et al.Three-dimensional structure and mimetic-membrane association of consensus 11-aminoacid motif from soybean LEA3 protein[J/OL].Peptide Science,2012,98(1):59-66[2018-01-01].https://doi.org/10.1002/bip.21693
[10]Chakrabortee S,Boschetti C,Walton L J,et al.Hydrophilic protein associated with desiccation tolerance exhibits broad protein stabilization function[J/OL].Proceedings of the National A-cademy of Sciences of the United States of America,2007,104(46):18073-18078[2018-01-01].https://doi.org/10.1073/pnas.0706964104
[11]Nakayama K,Okawa K,Kakizaki T,et al.Evaluation of the protective activities of a late embryogenesis abundant(LEA)related protein,Cor15am,during various stresses in vitro[J/OL].Bioscience,Biotechnology,and Biochemistry,2008,72(6):1642-1645[2018-01-01].https://doi.org/10.1271/bbb.80214
[12]Ried J L,Walker-Simmons M K.Group 3 Late Embryogenesis Abundant Proteins in desiccation-tolerant seedlings of wheat(Triticum aestivum L.)[J/OL].Plant Physiology,1993,102(1):125-131[2018-01-01].https://doi.org/10.1104/pp.102.1.125
[13]Moons A,Bauw G,Prinsen E,et al.Molecular and physiological responses to abscisic acid and salts in roots of salt-sensitive and salt-tolerant Indica rice varieties[J/OL].Plant Physiology,1995,107(1):177-186[2018-01-01].https://doi.org/10.1104/pp.107.1.177
[14]Ndong C,Danyluk J,Wilson K E,et al.Cold-regulated cereal chloroplast late embryogenesis abundant-like proteins.Molecular characterization and functional analyses[J/OL].Plant Physiology,2002,129(3):1368-1381[2018-01-01].https://doi.org/10.1104/pp.001925
[15]Xu Deping,Duan Xiaolan,Wang Baiyang,et al.Expression of a late embryogenesis abundant protein gene,HVA1,from barley confers tolerance to water deficit and salt stress in transgenic rice[J/OL].Plant Physiology,1996,110(1):249-257[2018-01-01].https://doi.org/10.1104/pp.110.1.249
[16]Yu Jianing,Zhang Jinsong,Shan Lun,et al.Two new group 3LEA genes of wheat and their functional analysis in yeast[J].Journal of Integrative Plant Biology,2005,47(11):1372-1381.
[17]王俊娟,穆敏,王帅,等.棉花脱水素GhDHN1的克隆及其表达[J].中国农业科学,2016,49(15):2867-2878.https://doi.org/10.3864/j.issn.0578-1752.2016.15.002Wang Junjuan,Mu Min,Wang Shuai,et al.Molecular clone and expression of GhDHN1 gene in cotton(Gossypium hirsutum L.)[J].Scientia Agricultura Sinica,2016,49(15):2867-2878.
[18]赵小洁,穆敏,陆许可,等.棉花耐盐相关基因Gh VP的表达及功能分析[J].棉花学报,2016,28(2):122-128.https://doi.org/10.11963/issn.1002-7807.201602004Zhao Xiaojie,Mu Min,Lu Xuke,et al.Expression and functional analysis of a salt tolerance-related gene from cotton[J].Cotton Science,2016,28(2):122-128.
[19]Afrin S,Zhu J,Cao H,et al.Molecular cloning and expression profile of an abiotic stress and hormone responsive MYB transcription factor gene from Panax ginseng[J/OL].Acta Biochimica et Biophysica Sinica,2015,47(4):267-277[2018-01-01].https://doi.org/10.1093/abbs/gmv012
[20]Clough S J,Bent A F.Floral dip:A simplified method for A-grobacterium-mediated transformation of Arabidopsis thaliana[J/OL].Plant Journal for Cell&Molecular Biology,1998,16(6):735-743[2018-01-01].https://doi.org/10.1046/j.1365-313x.1998.00343.x
[21]张天豹.棉花线粒体耐盐相关基因rps12、ccm C、nad的克隆及其表达分析[D].北京:中国农业科学院,2015.Zhang Tianbao.Clone and expression analysis of salt-tolerance related rps12,ccm C,nad gene in cotton mitochondria[D].Beijing:Chinese Academy of Agricultural Sciences,2015.
[22]胡根海,喻树迅.利用改良的CTAB法提取棉花叶片总RNA[J].棉花学报,2007,19(1):69-70.https://doi.org/10.3969/j.issn.1002-7807.2007.01.014Hu Genhai,Yu Shuxun.Extraction of high quality total RNA in cotton leaf with improved CTAB method[J].Cotton Science,2007,19(1):69-70.
[23]王俊娟,王帅,陆许可,等.棉花幼苗对低温胁迫的响应及抗冷机制初步研究[J].棉花学报,2017,29(2):147-156.https://doi.org/10.11963/issn.1002-7807.201702004Wang Junjuan,Wang Shuai,Lu Xueke,et al.The effect of low temperature stress on the growth of upland cotton seedlings and a preliminary study of cold-resistance mechanisms[J].Cotton Science,2017,29(2):147-156.
[24]Yang H P,Saitou T,Komeda Y,et al.Arabidopsis thaliana ECP63,encoding a LEA protein is located in chromosome 4[J/OL].Gene,1997,184(1):82-88[2018-01-01].https://doi.org/10.1016/S0378-1119(96)00578-1
[25]Franz G,Hatzopoulos P,Jones T J.Molecular and genetic analysis of an embryonic gene,DC8,from Daucus carota L.[J/OL].Molecular&General Genetics,1989,218(1):143-151[2018-01-01].https://doi.org/10.1007/bf00330577
[26]White C N,Rivin C J.Sequence and regulation of a late embryogenesis abundant group 3 protein of maize[J/OL].Plant Physiology,1995,108(3):1337-1338[2018-01-01].https://doi.org/10.1104/pp.108.3.1337
[27]Candat A,Paszkiewicz G,Neveu M,et al.The ubiquitous distribution of late embryogenesis abundant proteins across cell compartments in Arabidopsis offers tailored protection against abiotic stress[J/OL].Plant Cell,2014,26(7):3148-3166[2018-01-01].https://doi.org/10.1105/tpc.114.127316
[28]Garay-Arroyo A,Colmenero-Flores J M,Garciarrubio A,et al.Highly hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water deficit[J/OL].Journal of Biological Chemistry,2000,275(8):5668-5674[2018-01-01].https://doi.org/10.1074/jbc.275.8.5668
[29]Tunnacliffe A,Wise M J.The continuing conundrum of the LEA proteins[J/OL].Naturwissenschaften,2007,94(10):791-812[2018-01-01].https://doi.org/10.1007/s00114-007-0254-y
[30]Thalhammer A,Hundertmark M,Popova A V,et al.Interaction of two intrinsically disordered plant stress proteins(COR15Aand COR15B)with lipid membranes in the dry state[J/OL].Biochimica et Biophysica Acta,2010,1798(9):1812-1820[2018-01-01].https://doi.org/10.1016/j.bbamem.2010.05.015
[31]Ukaji N,Kuwabara C,Takezawa D,et al.Cold acclimation-induced WAP27 localized in endoplasmic reticulum in cortical parenchyma cells of mulberry tree was homologous to group 3late-embryogenesis abundant proteins[J/OL].Plant Physiology,2001,126(4):1588[2018-01-01].https://doi.org/10.1104/pp.126.4.1588
[32]Liu Yang,Liang Jianan,Sun Liping,et al.Group 3 LEA protein,ZmLEA3,is involved in protection from low temperature stress[J/OL].Frontiers in Plant Science,2016,7:1011[2018-01-01].https://doi.org/10.3389/fpls.2016.01011
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