蝴蝶兰PhTSJT1基因的克隆及在低温胁迫下的表达分析
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摘要
本研究从蝴蝶兰叶片中克隆了茎部特异基因Ph TSJT1的全长序列(GenBank登录号为MF797883),并分析了其在不同组织及低温胁迫下的表达特性。结果表明,Ph TSJT1基因全长994 bp,编码239个氨基酸,属于Class-Ⅱ谷氨酰胺酰胺基转移酶超家族成员;同源性分析表明,该蛋白与多种植物的茎部特异蛋白和铝诱导蛋白有较高的同源性,进化上与小兰屿蝴蝶兰的茎部特异蛋白亲缘关系最近;该基因在营养器官中表达水平较高,在花器官中表达水平较低;13℃/8℃(昼/夜)的低温胁迫抑制PhTSJT1基因的转录表达,并随着低温胁迫时间的延长,Ph TSJT1基因的表达水平逐渐降低,在温度恢复正常时其表达水平升高;4℃冷胁迫低温条件下,PhTSJT1基因在处理1 h时,表达水平升高,处理8 h时表达水平最高,16 h后表达水平逐渐降低。由此推测,PhTSJT1参与4℃冷胁迫的分子调控。本研究不但有助于理解热带亚热带植物的耐冷机制,也为蝴蝶兰新品种的遗传改良提供帮助。
The study cloned the full length sequence of stem specific gene PhTSJT1(GenBank accession number:MF797883) from the leaf of Phalaenopsis and analyzed the expression features under different issues and low temperature stress. The results showed the full-length sequence of PhTSJT1 gene was 994 bp, encoding 239 amino acids, which belonged to the superfamily of Class-Ⅱ glutamine transferases. The PhTSJT1 protein was highly homologous to stem-specific proteins and aluminum induced proteins from different species. Phylogenetic analysis indicated that the protein was closely related to stem-specific protein from Phalaenopsis equestris. The expression levels of PhTSJT1 gene were higher in vegetative organs than that of in floral organs. The transcriptional expression level of PhTSJT1 gene was inhibited with low temperature treatment of 13℃/8℃(day/night). With the extension of low-temperature stress time, the expression level of PhTSJT1 gene gradually decreased, then increased after the recovering to normal temperature. Under the cold stress of 4℃, the expressions of PhTSJT1 gene were increased at 1 h, and achieved the highest level at 8 h, of which the expression level was decreased gradually after treatment for 16 h. The results suggested that Ph TSJT1 participated in the molecular regulation of 4℃ cold stress.This study would be beneficial to understand the mechanism of cold tolerance in tropical and subtropical plant, as well as provide an aid for genetic improvement of new species in Phalaenopsis.
The study cloned the full length sequence of stem specific gene PhTSJT1(GenBank accession number:MF797883) from the leaf of Phalaenopsis and analyzed the expression features under different issues and low temperature stress. The results showed the full-length sequence of PhTSJT1 gene was 994 bp, encoding 239 amino acids, which belonged to the superfamily of Class-Ⅱ glutamine transferases. The PhTSJT1 protein was highly homologous to stem-specific proteins and aluminum induced proteins from different species. Phylogenetic analysis indicated that the protein was closely related to stem-specific protein from Phalaenopsis equestris. The expression levels of PhTSJT1 gene were higher in vegetative organs than that of in floral organs. The transcriptional expression level of PhTSJT1 gene was inhibited with low temperature treatment of 13℃/8℃(day/night). With the extension of low-temperature stress time, the expression level of PhTSJT1 gene gradually decreased, then increased after the recovering to normal temperature. Under the cold stress of 4℃, the expressions of PhTSJT1 gene were increased at 1 h, and achieved the highest level at 8 h, of which the expression level was decreased gradually after treatment for 16 h. The results suggested that Ph TSJT1 participated in the molecular regulation of 4℃ cold stress.This study would be beneficial to understand the mechanism of cold tolerance in tropical and subtropical plant, as well as provide an aid for genetic improvement of new species in Phalaenopsis.
引文
Alexandrov N.N.,Brover V.V.,Freidin S.,Troukhan M.E.,Tatarinova T.V.,Zhang H.,Swaller T.J.,Lu Y.P.,Bouck J.,Flavell R.B.,and Feldmann K.A.,2009,Insights into corn genes derived from large-scale cDNA sequencing,Plant Molecular Biology,69(1-2):179-194
Chen W.H.,Tseng Y.C.,Liu Y.C.,Chuo C.M.,Chen P.T.,Tseng K.M.,Yeh Y.C.,Ger M.J.,and Wang H.L.,2008,Coolnight temperature induces spike emergence and affects photosynthetic efficiency and metabolizable carbohydrate and organic acid pools in Phalaenopsis aphrodite,Plant Cell Reports.,27(10):1667-1675
Feng X.,Peng Z.Y.,Fu X.,Li S.,Zhang H.,Deng X.W.,and Fan L.M.,2012,Similarity and distinction between organ-specific gibberellin-modulated genome expression between Arabidopsis and rice,Environmental&Experimental Botany,78:127-137
Goodwin S.B.,and Sutter T.R.,2009,Microarray analysis of Arabidopsis genome response to aluminum stress,Biologia Plantarum,53(1):85-99
Guo W.J.,and Lee N.,2006,Effect of leaf and plant age and day/night temperature on net CO2uptake in Phalaenopsis amabilis var.Formosa,Journal of the American Society for Horticultural Science,131(3):320-326
Jiang Z.S.,Sun X.Q.,Ai X.Z.,Wang M.L.,Bi H.G.,and Wang H.T.,2010,Responses of rubisco and rubisco activase in cucumber seedlings to low temperature and weak light,Yingyong Shengtai Xuebao(Chinese Journal of Applied Ecology),21(8):2045-2050(姜振升,孙晓琦,艾希珍,王美玲,毕焕改,王洪涛,2010,低温弱光对黄瓜幼苗Rubisco与Rubisco活化酶的影响,应用生态学报,21(8):2045-2050)
Liang J.J.,2006,Transformation of Gassypium hirsutum Al-induced(GhAlin)gene,Thesis for M.S.,Xi'nan University,Supervisor:Pei Y.,pp.9-13(梁俊俊,2006,棉花铝诱导蛋白基因(Gh Alin)的遗传转化,硕士学位论文,西南大学,导师:裴炎,pp.9-13)
Liu F.,Vantoai T.,Moy L.P.,Bock G.,Linford L.D.,and Quackenbush J.,2005,Global transcription profiling reveals comprehensive insights into hypoxic response in Arabidopsis,Plant Physiology,137(3):1115-1129
Liu X.Q.,Wang X.F.,and Piao Y.J.,2007,A study on cold tolerance of different Phalaenopsis cultivars,Yuanyi Xuebao(Acta Horticulturae Sinica),34(2):425-430(刘学庆,王秀峰,朴永吉,2007,蝴蝶兰不同品种耐冷特性的研究,园艺学报,34(2):425-430)
Massière F.,and Badet-Denisot M.A.,1998,The mechanism of glutamine-dependent amidotransferases,Cellular Molecular Life Sciences,54(3):205-222
Nyunoya H.,and Lusty C.J.,1984,Sequence of the small subunit of yeast carbamyl phosphate synthetase and identification of its catalytic domain,Journal of Biological Chemistry,259(15):9790-9798
Ndong C.,Danyluk J.,Huner N.P.,and Sarhan F.,2001,Survey of gene expression in winter rye during changes in growth temperature,irradiance or excitation pressure,Plant Molecular Biology,45(6):691-703
Naidu S.L.,Moose S.P.,AL-Shoaibi A.K.,Raines C.A.,and Long S.P.,2003,Cold tolerance of C4 photosynthesis in Miscanthus×giganteus:adaptation in amounts and sequence of C4 photosynthetic enzymes,Plant Physiology,132(3):1688-1697
Richards K.D.,Snowden K.C.,and Gardner R.C.,1994,Wali6and wali7 genes induced by aluminum in wheat(Triticum aestivum L.)roots,Plant Physiology,105(4):1455-1456
Seurinck J.,Truettner J.,and Goldberg R.B.,1990,The nucleotide sequence of an anther-specific gene,Nucleic Acids Research,18(11):3403
Smith J.L.,1995,Structures of glutamine amidotransferases from the purine biosynthetic pathway,Biochem.Soc.Trans.,23(4):894-898
Siemens J.,Keller I.,Sarx J.,Kunz S.,Schuller A.,Nagel W.,Schmülling T.,Parniske M.,and Ludwig-Müller J.,2006,Transcriptome analysis of Arabidopsis clubroots indicate a key role for cytokinins in disease development,Molecular Plant Microbe Interactions,19(5):480-494
Weng M.L.,and Zalkin H.,1987,Structural role for a conserved region in the CTP synthetase glutamine amide transfer domain,Journal of Bacteriology,169(7):3023-3028
Xu S.P.,Zeng L.T.,and Ye Q.S.,2015,Effects of long-term elevated CO2on growth and flowering in Phalaenopsis,Yuanyi Xuebao(Acta Horticulturae Sinica),42(8):1599-1605(许申平,曾兰婷,叶庆生,2015,长期增施CO2对蝴蝶兰生长与开花的影响,园艺学报,42(8):1599-1605)
Yuan X.Y.,Liang F.,Jiang S.H.,Wan M.F.,Ma J.,Zhang X.Y.,and Cui B.,2015,Differential protein expression in phalaenopsis under low temperature,Applied Biochemistry Biotechnology,175(2):909-924
Zhao L.N.,Zhao Q.,Ao G.M.,and Yu J.J.,2009,The foxtail millet Si69 gene is a Wali7(wheat aluminum-induced protein 7)homologue and may function in aluminum tolerance,Chinese Science Bulletin,54(10):1697-1706
Zhang Z.,2014,Preliminary function analysis of AILP1 in Arabidopsis,Thesis for M.S.,Xi'nan University,Supervisor:Li D.M.,pp.2-5(张祚,2014,拟南芥AILP1基因功能的初步分析,硕士学位论文,西南大学,导师:李德蒙,pp.2-5)
Zhou S.,SauvéR.,and Thannhauser T.W.,2009,Proteome changes induced by aluminium stress in tomato roots,Journal of Experimental Botany,60(6):1849-1857
Chen W.H.,Tseng Y.C.,Liu Y.C.,Chuo C.M.,Chen P.T.,Tseng K.M.,Yeh Y.C.,Ger M.J.,and Wang H.L.,2008,Coolnight temperature induces spike emergence and affects photosynthetic efficiency and metabolizable carbohydrate and organic acid pools in Phalaenopsis aphrodite,Plant Cell Reports.,27(10):1667-1675
Feng X.,Peng Z.Y.,Fu X.,Li S.,Zhang H.,Deng X.W.,and Fan L.M.,2012,Similarity and distinction between organ-specific gibberellin-modulated genome expression between Arabidopsis and rice,Environmental&Experimental Botany,78:127-137
Goodwin S.B.,and Sutter T.R.,2009,Microarray analysis of Arabidopsis genome response to aluminum stress,Biologia Plantarum,53(1):85-99
Guo W.J.,and Lee N.,2006,Effect of leaf and plant age and day/night temperature on net CO2uptake in Phalaenopsis amabilis var.Formosa,Journal of the American Society for Horticultural Science,131(3):320-326
Jiang Z.S.,Sun X.Q.,Ai X.Z.,Wang M.L.,Bi H.G.,and Wang H.T.,2010,Responses of rubisco and rubisco activase in cucumber seedlings to low temperature and weak light,Yingyong Shengtai Xuebao(Chinese Journal of Applied Ecology),21(8):2045-2050(姜振升,孙晓琦,艾希珍,王美玲,毕焕改,王洪涛,2010,低温弱光对黄瓜幼苗Rubisco与Rubisco活化酶的影响,应用生态学报,21(8):2045-2050)
Liang J.J.,2006,Transformation of Gassypium hirsutum Al-induced(GhAlin)gene,Thesis for M.S.,Xi'nan University,Supervisor:Pei Y.,pp.9-13(梁俊俊,2006,棉花铝诱导蛋白基因(Gh Alin)的遗传转化,硕士学位论文,西南大学,导师:裴炎,pp.9-13)
Liu F.,Vantoai T.,Moy L.P.,Bock G.,Linford L.D.,and Quackenbush J.,2005,Global transcription profiling reveals comprehensive insights into hypoxic response in Arabidopsis,Plant Physiology,137(3):1115-1129
Liu X.Q.,Wang X.F.,and Piao Y.J.,2007,A study on cold tolerance of different Phalaenopsis cultivars,Yuanyi Xuebao(Acta Horticulturae Sinica),34(2):425-430(刘学庆,王秀峰,朴永吉,2007,蝴蝶兰不同品种耐冷特性的研究,园艺学报,34(2):425-430)
Massière F.,and Badet-Denisot M.A.,1998,The mechanism of glutamine-dependent amidotransferases,Cellular Molecular Life Sciences,54(3):205-222
Nyunoya H.,and Lusty C.J.,1984,Sequence of the small subunit of yeast carbamyl phosphate synthetase and identification of its catalytic domain,Journal of Biological Chemistry,259(15):9790-9798
Ndong C.,Danyluk J.,Huner N.P.,and Sarhan F.,2001,Survey of gene expression in winter rye during changes in growth temperature,irradiance or excitation pressure,Plant Molecular Biology,45(6):691-703
Naidu S.L.,Moose S.P.,AL-Shoaibi A.K.,Raines C.A.,and Long S.P.,2003,Cold tolerance of C4 photosynthesis in Miscanthus×giganteus:adaptation in amounts and sequence of C4 photosynthetic enzymes,Plant Physiology,132(3):1688-1697
Richards K.D.,Snowden K.C.,and Gardner R.C.,1994,Wali6and wali7 genes induced by aluminum in wheat(Triticum aestivum L.)roots,Plant Physiology,105(4):1455-1456
Seurinck J.,Truettner J.,and Goldberg R.B.,1990,The nucleotide sequence of an anther-specific gene,Nucleic Acids Research,18(11):3403
Smith J.L.,1995,Structures of glutamine amidotransferases from the purine biosynthetic pathway,Biochem.Soc.Trans.,23(4):894-898
Siemens J.,Keller I.,Sarx J.,Kunz S.,Schuller A.,Nagel W.,Schmülling T.,Parniske M.,and Ludwig-Müller J.,2006,Transcriptome analysis of Arabidopsis clubroots indicate a key role for cytokinins in disease development,Molecular Plant Microbe Interactions,19(5):480-494
Weng M.L.,and Zalkin H.,1987,Structural role for a conserved region in the CTP synthetase glutamine amide transfer domain,Journal of Bacteriology,169(7):3023-3028
Xu S.P.,Zeng L.T.,and Ye Q.S.,2015,Effects of long-term elevated CO2on growth and flowering in Phalaenopsis,Yuanyi Xuebao(Acta Horticulturae Sinica),42(8):1599-1605(许申平,曾兰婷,叶庆生,2015,长期增施CO2对蝴蝶兰生长与开花的影响,园艺学报,42(8):1599-1605)
Yuan X.Y.,Liang F.,Jiang S.H.,Wan M.F.,Ma J.,Zhang X.Y.,and Cui B.,2015,Differential protein expression in phalaenopsis under low temperature,Applied Biochemistry Biotechnology,175(2):909-924
Zhao L.N.,Zhao Q.,Ao G.M.,and Yu J.J.,2009,The foxtail millet Si69 gene is a Wali7(wheat aluminum-induced protein 7)homologue and may function in aluminum tolerance,Chinese Science Bulletin,54(10):1697-1706
Zhang Z.,2014,Preliminary function analysis of AILP1 in Arabidopsis,Thesis for M.S.,Xi'nan University,Supervisor:Li D.M.,pp.2-5(张祚,2014,拟南芥AILP1基因功能的初步分析,硕士学位论文,西南大学,导师:李德蒙,pp.2-5)
Zhou S.,SauvéR.,and Thannhauser T.W.,2009,Proteome changes induced by aluminium stress in tomato roots,Journal of Experimental Botany,60(6):1849-1857