梨糖转运相关基因PbTMT4启动子克隆及功能分析
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摘要
以‘砀山酥梨’(Pyrus bretschneideri‘Dangshan Suli’)为材料,利用梨基因组数据库,通过PCR获得了糖转运相关基因PbTMT4(Pbr032130.1)2 211 bp的CDS序列及其编码起始位点上游长度为1 220 bp的启动子序列。使用农杆菌介导法将PbTMT4导入拟南芥,与野生型对照植株相比,转基因拟南芥植株的生长速度更快,抽薹、开花时间更早,叶片糖积累量更高。生物信息学分析表明,该启动子中含有多个与逆境应答、激素信号和光信号相关的顺式作用元件。为进一步分析PbTMT4启动子功能,构建了该启动子与GUS基因融合的植物表达载体并转化拟南芥。对T_3代转基因拟南芥各组织进行GUS活性染色和半定量分析,发现GUS基因在根、茎、叶、花和果荚中均有表达,NaCl、干旱、GA_3、MeJA以及光照处理均能一定程度上提高转基因拟南芥中GUS基因的转录水平。逆境处理发现,PbTMT4转基因株系较野生型植株受到的伤害小。研究结果初步表明,PbTMT4可促进转基因拟南芥发育并提高糖积累量,可能在抗非生物胁迫中起重要的调控作用。
The 2 211 bp CDS sequence of PbTMT4 gene(Pbr032130.1)and the promoter sequence of 1 220 bp upstream of the transcription initiation site were obtained using Pyrus bretschneideri‘Dangshan Suli'as the material based on the pear genome database. When the Pb TMT4 gene was introduced into Arabidopsis by Agrobacterium-mediated method,the growth rate of transgenic Arabidopsis was faster,and the flowering time was earlier than the wild type. At the same time,the amount of sugar accumulation in transgenic Arabidopsis was significantly higher than that in wild type. Bioinformatics analysis indicated that the promoter contains multiple cis-acting elements associated with stress response,hormone signals and light signals. To further analyze the promoter function of PbTMT4 gene,a promoter-reporter vector was constructed and transformed into Arabidopsis. Analysis of T_3 transgenic Arabidopsis tissues with histochemical staining and semi-quantitative analysis showed that GUS gene was expressed in roots,stems,leaves,flowers and fruit pod. And sodium chloride(Na Cl),drought,gibberellin(GA_3),methyl jasmonate(MeJA)and light intensity can induce the transcription level of GUS gene in Arabidopsis to some extent. Stress treatment revealed that the PbTMT4 gene overexpression transgenic line had less damage than the wild type plants. The results of the study preliminarily suggest that PbTMT4 gene can promote the development of transgenic plants and increase sugar accumulation,and may play an important regulatory role in the abiotic stress.
The 2 211 bp CDS sequence of PbTMT4 gene(Pbr032130.1)and the promoter sequence of 1 220 bp upstream of the transcription initiation site were obtained using Pyrus bretschneideri‘Dangshan Suli'as the material based on the pear genome database. When the Pb TMT4 gene was introduced into Arabidopsis by Agrobacterium-mediated method,the growth rate of transgenic Arabidopsis was faster,and the flowering time was earlier than the wild type. At the same time,the amount of sugar accumulation in transgenic Arabidopsis was significantly higher than that in wild type. Bioinformatics analysis indicated that the promoter contains multiple cis-acting elements associated with stress response,hormone signals and light signals. To further analyze the promoter function of PbTMT4 gene,a promoter-reporter vector was constructed and transformed into Arabidopsis. Analysis of T_3 transgenic Arabidopsis tissues with histochemical staining and semi-quantitative analysis showed that GUS gene was expressed in roots,stems,leaves,flowers and fruit pod. And sodium chloride(Na Cl),drought,gibberellin(GA_3),methyl jasmonate(MeJA)and light intensity can induce the transcription level of GUS gene in Arabidopsis to some extent. Stress treatment revealed that the PbTMT4 gene overexpression transgenic line had less damage than the wild type plants. The results of the study preliminarily suggest that PbTMT4 gene can promote the development of transgenic plants and increase sugar accumulation,and may play an important regulatory role in the abiotic stress.
引文
Ayoubi T A,Wj V D V.1996.Regulation of gene expression by alternative promoters.Faseb Journal Official Publication of the Federation of American Societies for Experimental Biology,10(4):453-460.
Büttner M.2007.The monosaccharide transporter-(-like)gene family in Arabidopsis.FEBS Letters,581(12):2318-2324.
Cao Jian-dong,Chen Bai-hong,Wang Li-jun,Mao Juan,Zhao Xin.2010.Screening and evaluation of physiological indexes of cold resistance of grape.Northwest Botanical Journal,30(11):2232-2239.(in Chinese)曹建东,陈佰鸿,王利军,毛娟,赵鑫.2010.葡萄抗寒性生理指标筛选及其评价.西北植物学报,30(11):2232-2239.
Carninci P,Sandelin A,Lenhard B,Katayama S,Shimokawa K,Ponjavic J,Semple C A,Taylor M S,Engstr?m P G,Frith M C,Forrest A R,Alkema W B,Tan S L,Plessy C,Kodzius R,Ravasi T,Kasukawa T,Fukuda S,Kanamori-Katayama M,Kitazume Y,Kawaji H,Kai C,Nakamura M,Konno H,Nakano K,Mottagui-Tabar S,Arner P,Chesi A,Gustincich S,Persichetti F,Suzuki H,Grimmond S M,Wells C A,Orlando V,Wahlestedt C,Liu ET,Harbers M,Kawai J,Bajic V B,Hume D A,Hayashizaki Y.2006.Genome-wide analysis of mammalian promoter architecture and evolution.Nature Genetics,38(6):626-635.
Cheng R,Cheng Y S,LüJ H,Cheng J Q,Wang Y Z,Zhang S L,Zhang H P.2018.The gene PbTMT4 from pear(Pyrus bretschneideri)mediates vacuolar sugar transport and strongly affects sugar accumulation in fruit.Physiologia Plantarum,164(3):307-319.
Gu Ran,LIU Xiaofeng,Zhao Wensheng,Yan Shuangshuang,Sun Linhan,Wu Binning,Zhang Xiaolan.2018.Functional characterization of the promoter and second intron of cum1 during flower development in cucumber(Cucumis sativus L.).Horticultural Plant Journal,4(3):103-110.
Gutierrez L,Mauriat M,Guénin S,Pelloux J,Lefebvre J F,Louvet R,Rusterucci C,Moritz T,Guerineau F,Bellini C,Van W O.2008.The lack of a systematic validation of reference genes:a serious pitfall undervalued in reverse transcription-polymerase chain reaction(RT-PCR) analysis in plants.Plant Biotechnology Journal,6(6):609-618.
Kühn C,Frommer W B.1997.Macromolecular trafficking indicated by localization and turnover of sucrose transporters in enucleate sieve elements.Science,275(5304):1298-1300.
Lau O S,Deng X W.2010.Plant hormone signaling lightens up:integrators of light and hormones.Current Opinion in Plant Biology,13(5):571-577.
Li Fang,Shen Xin-jie,Zhao Kai,Yuan Hua-zhang,Li Tian-hong.2011.Cloning and functional analysis of MdSOT3 promoter of apple sorbitol transport gene.Acta Horticulturae Sinica,38(S):2457.(in Chinese)李芳,沈欣杰,赵凯,袁华招,李天红.2011.苹果山梨醇运输基因MdSOT3启动子克隆与功能分析.园艺学报,38(S):2457.
Li J M,Huang X S,Li L T,Zheng D M,Xue C,Zhang S L,Wu J.2015b.Proteome analysis of pear reveals key genes associated with fruit development and quality.Planta,241(6):1363-1379.
Li J M,Zheng D M,Li L T,Qiao X,Wei S W,Bai B,Zhang S L,Wu J.2015a.Genome-wide function,evolutionary characterization and expression analysis of sugar transporter family genes(Pyrus bretschneideri Rehd.).Plant&Cell Physiology,56(9):1721-1737.
Li X L,Xue C,Li J M,Qiao X,Li L,Yu L,Huang Y,Wu J.2016.Genome-wide identification,evolution and functional divergence of MYB transcription factors in Chinese white pear(Pyrus bretschneideri).Plant&Cell Physiology,57(4):824-847.
Li Y,Lee K K,Walsh S,Smith C,Hadingham S,Sorefan K,Bevan M W.2006.Establishing glucose-and ABA-regulated transcription networks in Arabidopsis by microarray analysis and promoter classification using a relevance vector machine.Genome Research,16(3):414-427.
Livak K J,Schmittgen T D.2012.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method.Methods,25(4):402-408.
Ma Xin-li,Qin Yuan,Wei Xiao-yu,Ma Feng-wang,Li Ming-jun.2014.Expression of TCT gene and its relationship with sugar accumulation.Acta Horticulturae Sinica,41(7),1317-1325.(in Chinese)马新立,秦源,魏晓钰,马锋旺,李明军.2014.苹果糖转运蛋白TMT基因的表达及其与糖积累的关系.园艺学报,41(7),1317-1325.
Marger M D,Jr S M.1993.A major superfamily of transmembrane facilitators that catalyse uniport,symport and antiport.Trends in Biochemical Sciences,18(1):13.Mamun E A,Alfred S,Cantrill L C,Overall R L,Sutton B G.2006.Effects of chillin gonmale gametophyte development in rice.Cell Biology International,30(7):583-591.
Martinoia E,Maeshima M,Neuhaus H E.2007.Vacuolar transporters and their essential role in plant metabolism.Journal of Experimental Botany,58(1):83-102.
Neuhaus H E.2007.Transport of primary metabolites across the plant vacuolar membrane.FEBS Letters,581(12):2223-2226.
Ni S,Meng L,Zhao J,Wang X,Chen J.2008.Isolation and characterization of the trichome-specific At TSG1 promoter from Arabidopsis thaliana.Plant Molecular Biology Reporter,26(4):263-276.
Nie Li-na,Xia Lan-qin,Xu Zhao-shi,Gao Dong-yao,Li Lin,Yu Zhuo,Chen Ming,Li Lian-cheng,Ma You-zhi.2008.Progress on cloning and functional study of plant gene promoter.Journal of Plant Genetic Resources,9(3):385-391.(in Chinese)聂丽娜,夏兰琴,徐兆师,高东尧,李琳,于卓,陈明,李连城,马有志.2008.植物基因启动子的克隆及其功能研究进展.植物遗传资源学报,9(3):385-391.
Pieterse C M J,Ton J,Loon L C V.2001.Cross-talk between plant defence signalling pathways:boost or burden.Agbiotechnet,3(1):8.
Schulz A,Beyhl D,Marten I,Wormit A,Neuhaus E,Poschet G,Büttner M,Schneider S,Sauer N,Hedrich R.2011.Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2.Plant Journal,68(1):129-136.
Taji T,Ohsumi C,Iuchi S,Seki M,Kasuga M,Kobayashi M.2002.Yamaguchi-Shinozaki K.,Shinozaki K.2010.Important roles of drought-and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana.Plant Journal,29(4):417-426.
Urao T,Shinozaki K.1993.An Arabidopsis MYB homolog is induced by dehydration stress and its gene product binds to the conserved MYBrecognition sequence.Plant Cell,5(11):1529-1539.
Wang L F,Qi X X,Huang X S,Xu L L,Jin C,Wu J,Zhang S L.2016.Overexpression of sucrose transporter gene PbSUT2 from Pyrus bretschneideri,enhances sucrose content in Solanum lycopersicum fruit.Plant Physiology&Biochemistry,105:150-161.
Wang Q,Yuan F,Pan Q F,Li M Y,Wang G F,Zhao J Y,Tang K.2010.Isolation and functional analysis of the Catharanthus roseus deacetylvindoline-4-o-acetyltransferase gene promoter.Plant Cell Reports,29(2):185-192.
Wingenter K,Schulz A,Wormit A,Wic S,Trentmann O,Imke I.2010.Increased activity of the vacuolar monosaccharide transporter tmt1 alters cellular sugar partitioning,sugar signaling,and seed yield in Arabidopsis.Plant Physiology,154(2):665-677.
Wormit A,Trentmann O,Feifer I,Lohr C,Tjaden J,Meyer S,Schmidt U,Martinoia E,Neuhaus H E.2006.Molecular identification and physiological characterization f a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport.Plant Cell,18(12):3476-3490.
Xu Haifeng,Qu Changzhi,Liu Jingxuan,Wang Yicheng,Wang Deyun,Zuo Weifang,Jiang Shenghui,Wang Nan,Zhang Zongying,Chen Xuesen.2017.Expression analysis and functional identification of a vacuolar sucrose transporter gene mdsut4 in apple.Acta Horticulturae Sinica,44(7):1235-1243.(in Chinese)许海峰,曲常志,刘静轩,王意程,王得云,左卫芳,姜生辉,王楠,张宗营,陈学森.2017.苹果液泡膜蔗糖转运蛋白基因MdSUT4的表达分析与功能鉴定.园艺学报,44(7):1235-1243.
Yamada K,Osakabe Y,Mizoi J,Nakashima K,Fujita Y,Shinozaki K,Yamaguchi-Shinozaki K.2010.Functional analysis of an Arabidopsis thaliana abiotic stress-inducible facilitated diffusion transporter for monosaccharides.Journal of Biological Chemistry,285(2):1138-1146.
Zhang X,Henriques R,Lin S S,Niu Q W,Chua N H.2006.Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method.Nature Protocols,1(2):641-646.
Zhao Jin,Sun Mei-hong,Hu Da-gang,Hao Yu-jin.2015.Molecular cloning and expression analysis of a hexokinase gene MdHXK1 in apple.Acta Horticulturae Sinica,42(8):1437-1447.(in Chinese)赵锦,孙美红,胡大刚,郝玉金.2015.苹果己糖激酶基因MdHXK1的克隆与表达分析.园艺学报,42(8):1437-1447.
Büttner M.2007.The monosaccharide transporter-(-like)gene family in Arabidopsis.FEBS Letters,581(12):2318-2324.
Cao Jian-dong,Chen Bai-hong,Wang Li-jun,Mao Juan,Zhao Xin.2010.Screening and evaluation of physiological indexes of cold resistance of grape.Northwest Botanical Journal,30(11):2232-2239.(in Chinese)曹建东,陈佰鸿,王利军,毛娟,赵鑫.2010.葡萄抗寒性生理指标筛选及其评价.西北植物学报,30(11):2232-2239.
Carninci P,Sandelin A,Lenhard B,Katayama S,Shimokawa K,Ponjavic J,Semple C A,Taylor M S,Engstr?m P G,Frith M C,Forrest A R,Alkema W B,Tan S L,Plessy C,Kodzius R,Ravasi T,Kasukawa T,Fukuda S,Kanamori-Katayama M,Kitazume Y,Kawaji H,Kai C,Nakamura M,Konno H,Nakano K,Mottagui-Tabar S,Arner P,Chesi A,Gustincich S,Persichetti F,Suzuki H,Grimmond S M,Wells C A,Orlando V,Wahlestedt C,Liu ET,Harbers M,Kawai J,Bajic V B,Hume D A,Hayashizaki Y.2006.Genome-wide analysis of mammalian promoter architecture and evolution.Nature Genetics,38(6):626-635.
Cheng R,Cheng Y S,LüJ H,Cheng J Q,Wang Y Z,Zhang S L,Zhang H P.2018.The gene PbTMT4 from pear(Pyrus bretschneideri)mediates vacuolar sugar transport and strongly affects sugar accumulation in fruit.Physiologia Plantarum,164(3):307-319.
Gu Ran,LIU Xiaofeng,Zhao Wensheng,Yan Shuangshuang,Sun Linhan,Wu Binning,Zhang Xiaolan.2018.Functional characterization of the promoter and second intron of cum1 during flower development in cucumber(Cucumis sativus L.).Horticultural Plant Journal,4(3):103-110.
Gutierrez L,Mauriat M,Guénin S,Pelloux J,Lefebvre J F,Louvet R,Rusterucci C,Moritz T,Guerineau F,Bellini C,Van W O.2008.The lack of a systematic validation of reference genes:a serious pitfall undervalued in reverse transcription-polymerase chain reaction(RT-PCR) analysis in plants.Plant Biotechnology Journal,6(6):609-618.
Kühn C,Frommer W B.1997.Macromolecular trafficking indicated by localization and turnover of sucrose transporters in enucleate sieve elements.Science,275(5304):1298-1300.
Lau O S,Deng X W.2010.Plant hormone signaling lightens up:integrators of light and hormones.Current Opinion in Plant Biology,13(5):571-577.
Li Fang,Shen Xin-jie,Zhao Kai,Yuan Hua-zhang,Li Tian-hong.2011.Cloning and functional analysis of MdSOT3 promoter of apple sorbitol transport gene.Acta Horticulturae Sinica,38(S):2457.(in Chinese)李芳,沈欣杰,赵凯,袁华招,李天红.2011.苹果山梨醇运输基因MdSOT3启动子克隆与功能分析.园艺学报,38(S):2457.
Li J M,Huang X S,Li L T,Zheng D M,Xue C,Zhang S L,Wu J.2015b.Proteome analysis of pear reveals key genes associated with fruit development and quality.Planta,241(6):1363-1379.
Li J M,Zheng D M,Li L T,Qiao X,Wei S W,Bai B,Zhang S L,Wu J.2015a.Genome-wide function,evolutionary characterization and expression analysis of sugar transporter family genes(Pyrus bretschneideri Rehd.).Plant&Cell Physiology,56(9):1721-1737.
Li X L,Xue C,Li J M,Qiao X,Li L,Yu L,Huang Y,Wu J.2016.Genome-wide identification,evolution and functional divergence of MYB transcription factors in Chinese white pear(Pyrus bretschneideri).Plant&Cell Physiology,57(4):824-847.
Li Y,Lee K K,Walsh S,Smith C,Hadingham S,Sorefan K,Bevan M W.2006.Establishing glucose-and ABA-regulated transcription networks in Arabidopsis by microarray analysis and promoter classification using a relevance vector machine.Genome Research,16(3):414-427.
Livak K J,Schmittgen T D.2012.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method.Methods,25(4):402-408.
Ma Xin-li,Qin Yuan,Wei Xiao-yu,Ma Feng-wang,Li Ming-jun.2014.Expression of TCT gene and its relationship with sugar accumulation.Acta Horticulturae Sinica,41(7),1317-1325.(in Chinese)马新立,秦源,魏晓钰,马锋旺,李明军.2014.苹果糖转运蛋白TMT基因的表达及其与糖积累的关系.园艺学报,41(7),1317-1325.
Marger M D,Jr S M.1993.A major superfamily of transmembrane facilitators that catalyse uniport,symport and antiport.Trends in Biochemical Sciences,18(1):13.Mamun E A,Alfred S,Cantrill L C,Overall R L,Sutton B G.2006.Effects of chillin gonmale gametophyte development in rice.Cell Biology International,30(7):583-591.
Martinoia E,Maeshima M,Neuhaus H E.2007.Vacuolar transporters and their essential role in plant metabolism.Journal of Experimental Botany,58(1):83-102.
Neuhaus H E.2007.Transport of primary metabolites across the plant vacuolar membrane.FEBS Letters,581(12):2223-2226.
Ni S,Meng L,Zhao J,Wang X,Chen J.2008.Isolation and characterization of the trichome-specific At TSG1 promoter from Arabidopsis thaliana.Plant Molecular Biology Reporter,26(4):263-276.
Nie Li-na,Xia Lan-qin,Xu Zhao-shi,Gao Dong-yao,Li Lin,Yu Zhuo,Chen Ming,Li Lian-cheng,Ma You-zhi.2008.Progress on cloning and functional study of plant gene promoter.Journal of Plant Genetic Resources,9(3):385-391.(in Chinese)聂丽娜,夏兰琴,徐兆师,高东尧,李琳,于卓,陈明,李连城,马有志.2008.植物基因启动子的克隆及其功能研究进展.植物遗传资源学报,9(3):385-391.
Pieterse C M J,Ton J,Loon L C V.2001.Cross-talk between plant defence signalling pathways:boost or burden.Agbiotechnet,3(1):8.
Schulz A,Beyhl D,Marten I,Wormit A,Neuhaus E,Poschet G,Büttner M,Schneider S,Sauer N,Hedrich R.2011.Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2.Plant Journal,68(1):129-136.
Taji T,Ohsumi C,Iuchi S,Seki M,Kasuga M,Kobayashi M.2002.Yamaguchi-Shinozaki K.,Shinozaki K.2010.Important roles of drought-and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana.Plant Journal,29(4):417-426.
Urao T,Shinozaki K.1993.An Arabidopsis MYB homolog is induced by dehydration stress and its gene product binds to the conserved MYBrecognition sequence.Plant Cell,5(11):1529-1539.
Wang L F,Qi X X,Huang X S,Xu L L,Jin C,Wu J,Zhang S L.2016.Overexpression of sucrose transporter gene PbSUT2 from Pyrus bretschneideri,enhances sucrose content in Solanum lycopersicum fruit.Plant Physiology&Biochemistry,105:150-161.
Wang Q,Yuan F,Pan Q F,Li M Y,Wang G F,Zhao J Y,Tang K.2010.Isolation and functional analysis of the Catharanthus roseus deacetylvindoline-4-o-acetyltransferase gene promoter.Plant Cell Reports,29(2):185-192.
Wingenter K,Schulz A,Wormit A,Wic S,Trentmann O,Imke I.2010.Increased activity of the vacuolar monosaccharide transporter tmt1 alters cellular sugar partitioning,sugar signaling,and seed yield in Arabidopsis.Plant Physiology,154(2):665-677.
Wormit A,Trentmann O,Feifer I,Lohr C,Tjaden J,Meyer S,Schmidt U,Martinoia E,Neuhaus H E.2006.Molecular identification and physiological characterization f a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport.Plant Cell,18(12):3476-3490.
Xu Haifeng,Qu Changzhi,Liu Jingxuan,Wang Yicheng,Wang Deyun,Zuo Weifang,Jiang Shenghui,Wang Nan,Zhang Zongying,Chen Xuesen.2017.Expression analysis and functional identification of a vacuolar sucrose transporter gene mdsut4 in apple.Acta Horticulturae Sinica,44(7):1235-1243.(in Chinese)许海峰,曲常志,刘静轩,王意程,王得云,左卫芳,姜生辉,王楠,张宗营,陈学森.2017.苹果液泡膜蔗糖转运蛋白基因MdSUT4的表达分析与功能鉴定.园艺学报,44(7):1235-1243.
Yamada K,Osakabe Y,Mizoi J,Nakashima K,Fujita Y,Shinozaki K,Yamaguchi-Shinozaki K.2010.Functional analysis of an Arabidopsis thaliana abiotic stress-inducible facilitated diffusion transporter for monosaccharides.Journal of Biological Chemistry,285(2):1138-1146.
Zhang X,Henriques R,Lin S S,Niu Q W,Chua N H.2006.Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method.Nature Protocols,1(2):641-646.
Zhao Jin,Sun Mei-hong,Hu Da-gang,Hao Yu-jin.2015.Molecular cloning and expression analysis of a hexokinase gene MdHXK1 in apple.Acta Horticulturae Sinica,42(8):1437-1447.(in Chinese)赵锦,孙美红,胡大刚,郝玉金.2015.苹果己糖激酶基因MdHXK1的克隆与表达分析.园艺学报,42(8):1437-1447.