番茄果实成熟过程中SlSWEET7a的功能分析
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摘要
【目的】SWEETs(sugars will eventually be exported transporters)是一种糖转运蛋白,参与植物生物进程,对植物生长发育、响应各种胁迫、宿主-病原体的互作发挥作用。克隆番茄SWEET7a,通过构建Sl SWEET7a沉默和过表达载体,研究其在糖的转运过程中的作用,为探索SWEETs在植物果实发育过程中的功能提供理论依据。【方法】以Micro-Tom(Solanum lycopersicum)番茄为试材,利用RT-PCR技术从果实中克隆SWEET7a的c DNA全长842bp,进行生物信息学分析,并利用MEGA6.0构建拟南芥进化树,与Sl SWEET7a进行蛋白序列同源性分析;利用实时荧光定量PCR技术探明其在果实发育时期的时空表达特征分析,并构建基因的沉默和过表达载体,通过农杆菌介导的果实注射法进行瞬时表达检测构建载体的表达效率;然后进行番茄的遗传转化,获得T1代转基因株系,利用实时荧光定量PCR技术检测绿熟期果实SWEET7a的表达,通过高效液相色谱法检测转基因果实和叶片中糖组成与含量的变化。【结果】Sl SWEET7a蛋白结构是由7个跨膜结构域构成的。同源性比对分析结果显示,Sl SWEET7a与拟南芥At SWEET6和At SWEET8序列同源性较高,都属于SWEETs家族的CladeⅡ。番茄果实各部位的表达分析显示,Sl SWEET7a在绿熟期果柄、果实维管束相对表达量最高,转色期和红熟期相对表达量较低。构建SWEET7a沉默(S7a)及过表达载体(OE7a)在番茄果实的瞬时表达,发现OE7a样品果实中Sl SWEET7a的表达量是未注射果实的6倍,其Sl SWEET7a表达量明显上调,与对照相比,S7a样品果实中Sl SWEET7a明显下调了5倍。在番茄中的遗传转化中卡那霉素抗性筛选获得10株可能的超表达T0代植株,PCR鉴定得到了Sl SWEET7a超表达8株;沉默株系经除草剂筛选,获得14株,PCR检测得到10株沉默株系。T1代植株的实时定量分析显示,过表达Sl SWEET7a植株发生转基因沉默现象,Sl SWEET7a表达量显著低于正常植株,而沉默植株表达量也降低,说明获得的过表达植株也发生了基因沉默。果糖、葡萄糖和蔗糖含量测定结果表明,降低番茄中Sl SWEET7a的表达,植株成熟叶片和绿熟期果实中果糖、葡萄糖和蔗糖含量均高于对照,尤其是叶片中蔗糖含量显著高于对照,这说明Sl SWEET7a对细胞中蔗糖的易化扩散起着重要作用。【结论】Sl SWEET7a对叶片中蔗糖向源组织韧皮部的装载及果实果柄、维管束的运输、卸载起重要调控作用。
【Objective】 The SWEETs(Sugars Will Eventually Be Exported Transporters) is a kind of sugar transporters which are involved in plant biological processes and play key roles in plant growth and development in response to various stresses and host-pathogen interaction. SWEET7 a was cloned and its function was analyzed to provide the theoretical foundation for exploring the function of SWEETs during fruit development in plant by constructing the silence and overexpression of Sl SWEET7 a.【Method】Using Micro-Tom(Solanum lycopersicum) as a test material, the 842 bp full-length Sl SWEET7 a c DNA was cloned in fruits. The phylogenetic tree of SWEET proteins from Arabidopsis was constructed by using MEGA6.0, and homology of Sl SWEET7 a protein sequences was analysed compared with Arabidopsis. RT-PCR was performed to analyse the spatiotemporal expression of Sl Sweet7 a during the fruits development periods. The Sl SWEET7 a silence vector and overexpression vector were constructed and transformed into tomato by Agrobacterium-mediated genetic transformation. The efficiency of two vectors was examined by the transient expression of Agrobacterium tumefaciens injection method. The expressions of SWEET7 a in T1 generation transgenic green fruits were studied by quantitative PCR. The changes of sugar composition and content in transgenic fruits and leaves were detected by high performance liquid chromatography.【Result】The bioinformatics analysis of protein sequence showed that the Sl SWEET7 a is consisted of seven transmembrane domains. Sl SWEET7 a belongs to the CladeⅡof the SWEETs gene family, which was highly homologous to At SWEET6 and At SWEET8 in Arabidopsis thaliana. The analysis of spatiotemporal expression indicated that Sl SWEET7 a expression level was the highest at stalks and vascular bundles of mature green stage in tomato fruits, while the relative expression level was lower in breaker fruits and red ripping stage. The transient expression of SWEET7 a silencing(S7 a) and overexpression(OE7 a) vector in tomato fruits was observed. The expression level of Sl SWEET7 a in fruit of OE7 a was 6 times higher than that of non-injected fruit, which was significantly up-regulated compared with control. However, the expression level of Sl SWEET7 a in S7 a was significantly decreased 5 times. Ten overexpression lines were obtained by kanamycin resistance screening, and eight overexpression lines were obtained by PCR. Fourteen silencing lines were screened by phosphinothricin and 10 transgenic silence lines were obtained by PCR. Real-time quantitative PCR analysis of the SWEET7 a expression in T1 generation lines revealed that gene silencing occurred in overexpressed plants. The expression level of Sl SWEET7 a-overexpressing transgenic plants was significantly lower than that in wild plants, so did the silencing plants. Those results explained that the overexpressing plants also had the phenomenon of gene silencing. The determination of contents of fructose, glucose, sucrose showed that the silencing and overexpressing plants were higher than that of the wild type in the leaves and fruits after reducing expression of Sl SWEET7 a in tomato. Especially, the sucrose content of leaves was significantly increased. This showed that Sl SWEET7 a plays an important role in the facilitated diffusion of sucrose in cells.【Conclusion】Sl SWEET7 a plays an important role in regulating the loading of sucrose into the phloem of fruit tissue, the transportation and unloading of fruit stalks and vascular bundles.
【Objective】 The SWEETs(Sugars Will Eventually Be Exported Transporters) is a kind of sugar transporters which are involved in plant biological processes and play key roles in plant growth and development in response to various stresses and host-pathogen interaction. SWEET7 a was cloned and its function was analyzed to provide the theoretical foundation for exploring the function of SWEETs during fruit development in plant by constructing the silence and overexpression of Sl SWEET7 a.【Method】Using Micro-Tom(Solanum lycopersicum) as a test material, the 842 bp full-length Sl SWEET7 a c DNA was cloned in fruits. The phylogenetic tree of SWEET proteins from Arabidopsis was constructed by using MEGA6.0, and homology of Sl SWEET7 a protein sequences was analysed compared with Arabidopsis. RT-PCR was performed to analyse the spatiotemporal expression of Sl Sweet7 a during the fruits development periods. The Sl SWEET7 a silence vector and overexpression vector were constructed and transformed into tomato by Agrobacterium-mediated genetic transformation. The efficiency of two vectors was examined by the transient expression of Agrobacterium tumefaciens injection method. The expressions of SWEET7 a in T1 generation transgenic green fruits were studied by quantitative PCR. The changes of sugar composition and content in transgenic fruits and leaves were detected by high performance liquid chromatography.【Result】The bioinformatics analysis of protein sequence showed that the Sl SWEET7 a is consisted of seven transmembrane domains. Sl SWEET7 a belongs to the CladeⅡof the SWEETs gene family, which was highly homologous to At SWEET6 and At SWEET8 in Arabidopsis thaliana. The analysis of spatiotemporal expression indicated that Sl SWEET7 a expression level was the highest at stalks and vascular bundles of mature green stage in tomato fruits, while the relative expression level was lower in breaker fruits and red ripping stage. The transient expression of SWEET7 a silencing(S7 a) and overexpression(OE7 a) vector in tomato fruits was observed. The expression level of Sl SWEET7 a in fruit of OE7 a was 6 times higher than that of non-injected fruit, which was significantly up-regulated compared with control. However, the expression level of Sl SWEET7 a in S7 a was significantly decreased 5 times. Ten overexpression lines were obtained by kanamycin resistance screening, and eight overexpression lines were obtained by PCR. Fourteen silencing lines were screened by phosphinothricin and 10 transgenic silence lines were obtained by PCR. Real-time quantitative PCR analysis of the SWEET7 a expression in T1 generation lines revealed that gene silencing occurred in overexpressed plants. The expression level of Sl SWEET7 a-overexpressing transgenic plants was significantly lower than that in wild plants, so did the silencing plants. Those results explained that the overexpressing plants also had the phenomenon of gene silencing. The determination of contents of fructose, glucose, sucrose showed that the silencing and overexpressing plants were higher than that of the wild type in the leaves and fruits after reducing expression of Sl SWEET7 a in tomato. Especially, the sucrose content of leaves was significantly increased. This showed that Sl SWEET7 a plays an important role in the facilitated diffusion of sucrose in cells.【Conclusion】Sl SWEET7 a plays an important role in regulating the loading of sucrose into the phloem of fruit tissue, the transportation and unloading of fruit stalks and vascular bundles.
引文
[1]ROITSCH T,GONZALEZ M C.Function and regulation of plant invertases:Sweet sensations.Trends in Plant Science,2004,9:606-613.
[2]GUO W J,NAGY R,CHEN H Y,PFRUNDER S,YU Y C,SANTELIA D,MARTINOIA E.SWEET17,a facilitative transporter,mediates fructose transport across the tonoplast of Arabidopsis roots and leaves.Plant Physiology,2014,164(2):777-789.
[3]LEMOINE R,LA CAMERA S,ATANASSOVA R,DéDALDéCHAMP F,ALLARIO T,POURTAU N,FAUCHER M.Source-to-sink transport of sugar and regulation by environmental factors.Frontiers in Plant Science,2013,4:272.
[4]CHEN L Q,HOU B H,LALONDE S,TAKANAGA H,HARTUNG M L,QU X Q,CHERMAK D.Sugar transporters for intercellular exchange and nutrition of pathogens.Nature,2010,468:527-532.
[5]BRAUN D M.SWEET!The pathway is complete.Science,2012,335(6065):173-174.
[6]FENG C Y,HAN J X,HAN X X,JIANG J.Genome-wide identification,phylogeny,and expression analysis of the SWEET gene family in tomato.Gene,2015,573(2):261-272.
[7]CHEN L Q,QU X Q,HOU B H,SOSSO D,OSORIO S,FERNIE A R,Frommer W B.Sucrose efflux mediated by SWEET proteins as a key step for phloem transport.Science,2012,335:207–211.
[8]EOM J S,CHEN L Q,SOSSO D,JULIUS B T,LIN I W,QU X Q,FROMMER W B.SWEETs,transporters for intracellular and intercellular sugar translocation.Current Opinion in Plant Biology,2015,25:53-62.
[9]ENGEL M L,DAVIS R H,MCCORMICK S,MCCORMICK S.Green sperm.Identification of male gamete promoters in Arabidopsis.Plant Physiology,2005,138:2124-2133.
[10]GUAN Y F,HUANG X Y,ZHU J,GAO J F,ZHANG H X,YANG Z N,YANG Z N.RUPTURED POLLEN GRAIN1,a member of the Mt N3/saliva gene family,is crucial for exine pattern formation and cell integrity of microspores in Arabidopsis.Plant Physiology,2008,147:852-863.
[11]SUN M X,HUANG X Y,YANG J,GUAN Y F,YANG Z N.Arabidopsis RPG1 is important for primexine deposition and functions redundantly with RPG2 for plant fertility at the late reproductive stage.Plant Reproduction,2013,26,83-91.
[12]MORIYAMA E N,STROPE P K,OPIYO S O,CHEN Z,JONES A M.Mining the Arabidopsis thaliana genome for highly-divergent seven transmembrane receptors.Genome Biology,2006,7(10):R96.
[13]KANN Y,OIKAWA T,CHIBA Y,ISHIMARU Y,SHIMIZU T,SANO N,SEO M.At SWEET13 and At SWEET14 regulate gibberellin-mediated physiological processes.Nature Communications,2016,7:13245.
[14]YUAN M,WANG S.Rice Mt N3/saliva/SWEET family genes and their homologs in cellular organisms.Molecular Plant,2013,6(3):665-674.
[15]CHEN L Q,LIN I W,QU X Q,SOSSO D,MCFARLANE H E,LONDONO A,SAMUELS A L,FROMMER W B,FROMMER W B.A cascade of sequentially expressed sucrose transporters in the seed coat and endosperm provides nutrition for the Arabidopsis embryo.The Plant Cell,2015,27:607-619.
[16]MA L,ZHANG D,MIAO Q,YANG J,XUAN Y,HU Y.Essential role of sugar transporter Os SWEET11 during the early stage of rice grain filling.Plant and Cell Physiology,2017,58(5):863-873.
[17]CHU Z,FU B,YANG H,XU C,LI Z,SANCHEZ A,WANG S.Targeting xa13,a recessive gene for bacterial blight resistance in rice.Theoretical and Applied Genetics,2006,112(3):455-461.
[18]SOSSO D,LUO D,LI Q B,SASSE J,YANG J,GENDROT G,ROGOWSKY P M.Seed filling in domesticated maize and rice depends on SWEET-mediated hexose transport.Nature genetics,2015,47(12):1489-1493.
[19]LIU X,ZHANG Y,YANG C,TIAN Z,LI J.At SWEET4,a hexose facilitator,mediates sugar transport to axial sinks and affects plant development.Scientific Reports,2016,6:24563.
[20]ZHOU Y,LIU L,HUANG W,YUAN M,ZHOU F,LI X,LIN Y.Overexpression of Os SWEET5 in rice causes growth retardation and precocious senescence.PLo S ONE,2014,9(4):e94210.
[21]GUO M,ZHANG Y L,MENG Z J,JIANG J.Optimization of factors affecting Agrobacterium-mediated transformation of Micro-Tom tomatoes.Genetics and Molecular Research,2012,11(1):661-671.
[22]郝敬虹,李天来,孟思达,赵博,孙利萍.夜间低温对薄皮甜瓜果实糖积累及代谢相关酶活性的影响.中国农业科学,2009,42(10):3592-3599.HAO J H,LI T L,MENG S D,ZHAO B,SUN L P.Effects of night low temperature on sugar accumulation and sugar-metabolizing enzyme activities in melon fruit.Scientia Agricultura Sinica,2009,42(10):3592-3599.(in Chinese)
[23]WELLMER F,ALVES-FERREIRA M,DUBOIS A,RIECHMANN J L,MEYEROWITZ E M.Genome-wide analysis of gene expression during early Arabidopsis flower development.PLo S Genetics,2006,2(7):e117.
[24]BOCK K W,HONYS D,WARD J M,PADMANABAN S,NAWROCKI E P,HIRSCHI K D,SZE H.Integrating membrane transport with male gametophyte development and function through transcriptomics.Plant Physiology,2006,140(4):1151-1168.
[25]刘畅,姜晶,韩晓雪,韩佳轩.植物中SWEET基因家族研究进展.植物生理学报,2014,509:1367-1373.LIU C,JIANG J,HAN X X,HAN J X.Research advances in SWEET gene family in plants.Plant Physiology Journal,2014,509:1367-1373.(in Chinese)
[26]CHONG J,PIRON M C,MEYER S,MEYER S,MERDINOGLU D,BERTSCH C,MESTRE P.The SWEET family of sugar transporters in grapevine:Vv SWEET4 is involved in the interaction with Botrytis cinerea.Journal of Experimental Botany,2014,65(22):6589-6601.
[27]赵文婷,魏建和,刘晓东,高志晖.植物瞬时表达技术的主要方法与应用进展.生物技术通讯,2013,2:294-300.ZHAO W T,WEI J H,LIU X D,GAO Z H.Advance of the main methods and applications of plant transient expression system.Letters in Biotechnology,2013,2:294-300.(in Chinese)
[28]常丽娟,刘勇,宋君,刘文娟,张富丽,王东,尹全,雷绍荣.转基因植物中外源基因的沉默及应对策略.生物技术通讯,2013,24(6):881-885.CHANG L J,LIU Y,SONG J,LIU W J,ZHANG F L,WANG D,YIN Q,LEI S R.Silence of exogenous genes in transgenic plants and the coping strategies.Letters in Biotechnology,2013,24(6):881-885.(in Chinese)
[29]YUAN M,ZHAO J,HUANG R,LI X,XIAO J,WANG S.Rice Mt N3/saliva/SWEET gene family:Evolution,expression profiling,and sugar transport.Journal of Integrative Plant Biology,2014,56(6):559-570.
[30]BüTTNER M,SAUER N.Monosaccharide transporters in plants:Structure,function and physiology.Biochimica et Biophysica Acta(BBA)-Biomembranes,2000,1465(1):263-274.
[31]LALONDE S,WIPF D,FROMMER W B.Transport mechanisms for organic forms of carbon and nitrogen between source and sink.Annual Review of Plant Biology,2004,55:341-372.
[2]GUO W J,NAGY R,CHEN H Y,PFRUNDER S,YU Y C,SANTELIA D,MARTINOIA E.SWEET17,a facilitative transporter,mediates fructose transport across the tonoplast of Arabidopsis roots and leaves.Plant Physiology,2014,164(2):777-789.
[3]LEMOINE R,LA CAMERA S,ATANASSOVA R,DéDALDéCHAMP F,ALLARIO T,POURTAU N,FAUCHER M.Source-to-sink transport of sugar and regulation by environmental factors.Frontiers in Plant Science,2013,4:272.
[4]CHEN L Q,HOU B H,LALONDE S,TAKANAGA H,HARTUNG M L,QU X Q,CHERMAK D.Sugar transporters for intercellular exchange and nutrition of pathogens.Nature,2010,468:527-532.
[5]BRAUN D M.SWEET!The pathway is complete.Science,2012,335(6065):173-174.
[6]FENG C Y,HAN J X,HAN X X,JIANG J.Genome-wide identification,phylogeny,and expression analysis of the SWEET gene family in tomato.Gene,2015,573(2):261-272.
[7]CHEN L Q,QU X Q,HOU B H,SOSSO D,OSORIO S,FERNIE A R,Frommer W B.Sucrose efflux mediated by SWEET proteins as a key step for phloem transport.Science,2012,335:207–211.
[8]EOM J S,CHEN L Q,SOSSO D,JULIUS B T,LIN I W,QU X Q,FROMMER W B.SWEETs,transporters for intracellular and intercellular sugar translocation.Current Opinion in Plant Biology,2015,25:53-62.
[9]ENGEL M L,DAVIS R H,MCCORMICK S,MCCORMICK S.Green sperm.Identification of male gamete promoters in Arabidopsis.Plant Physiology,2005,138:2124-2133.
[10]GUAN Y F,HUANG X Y,ZHU J,GAO J F,ZHANG H X,YANG Z N,YANG Z N.RUPTURED POLLEN GRAIN1,a member of the Mt N3/saliva gene family,is crucial for exine pattern formation and cell integrity of microspores in Arabidopsis.Plant Physiology,2008,147:852-863.
[11]SUN M X,HUANG X Y,YANG J,GUAN Y F,YANG Z N.Arabidopsis RPG1 is important for primexine deposition and functions redundantly with RPG2 for plant fertility at the late reproductive stage.Plant Reproduction,2013,26,83-91.
[12]MORIYAMA E N,STROPE P K,OPIYO S O,CHEN Z,JONES A M.Mining the Arabidopsis thaliana genome for highly-divergent seven transmembrane receptors.Genome Biology,2006,7(10):R96.
[13]KANN Y,OIKAWA T,CHIBA Y,ISHIMARU Y,SHIMIZU T,SANO N,SEO M.At SWEET13 and At SWEET14 regulate gibberellin-mediated physiological processes.Nature Communications,2016,7:13245.
[14]YUAN M,WANG S.Rice Mt N3/saliva/SWEET family genes and their homologs in cellular organisms.Molecular Plant,2013,6(3):665-674.
[15]CHEN L Q,LIN I W,QU X Q,SOSSO D,MCFARLANE H E,LONDONO A,SAMUELS A L,FROMMER W B,FROMMER W B.A cascade of sequentially expressed sucrose transporters in the seed coat and endosperm provides nutrition for the Arabidopsis embryo.The Plant Cell,2015,27:607-619.
[16]MA L,ZHANG D,MIAO Q,YANG J,XUAN Y,HU Y.Essential role of sugar transporter Os SWEET11 during the early stage of rice grain filling.Plant and Cell Physiology,2017,58(5):863-873.
[17]CHU Z,FU B,YANG H,XU C,LI Z,SANCHEZ A,WANG S.Targeting xa13,a recessive gene for bacterial blight resistance in rice.Theoretical and Applied Genetics,2006,112(3):455-461.
[18]SOSSO D,LUO D,LI Q B,SASSE J,YANG J,GENDROT G,ROGOWSKY P M.Seed filling in domesticated maize and rice depends on SWEET-mediated hexose transport.Nature genetics,2015,47(12):1489-1493.
[19]LIU X,ZHANG Y,YANG C,TIAN Z,LI J.At SWEET4,a hexose facilitator,mediates sugar transport to axial sinks and affects plant development.Scientific Reports,2016,6:24563.
[20]ZHOU Y,LIU L,HUANG W,YUAN M,ZHOU F,LI X,LIN Y.Overexpression of Os SWEET5 in rice causes growth retardation and precocious senescence.PLo S ONE,2014,9(4):e94210.
[21]GUO M,ZHANG Y L,MENG Z J,JIANG J.Optimization of factors affecting Agrobacterium-mediated transformation of Micro-Tom tomatoes.Genetics and Molecular Research,2012,11(1):661-671.
[22]郝敬虹,李天来,孟思达,赵博,孙利萍.夜间低温对薄皮甜瓜果实糖积累及代谢相关酶活性的影响.中国农业科学,2009,42(10):3592-3599.HAO J H,LI T L,MENG S D,ZHAO B,SUN L P.Effects of night low temperature on sugar accumulation and sugar-metabolizing enzyme activities in melon fruit.Scientia Agricultura Sinica,2009,42(10):3592-3599.(in Chinese)
[23]WELLMER F,ALVES-FERREIRA M,DUBOIS A,RIECHMANN J L,MEYEROWITZ E M.Genome-wide analysis of gene expression during early Arabidopsis flower development.PLo S Genetics,2006,2(7):e117.
[24]BOCK K W,HONYS D,WARD J M,PADMANABAN S,NAWROCKI E P,HIRSCHI K D,SZE H.Integrating membrane transport with male gametophyte development and function through transcriptomics.Plant Physiology,2006,140(4):1151-1168.
[25]刘畅,姜晶,韩晓雪,韩佳轩.植物中SWEET基因家族研究进展.植物生理学报,2014,509:1367-1373.LIU C,JIANG J,HAN X X,HAN J X.Research advances in SWEET gene family in plants.Plant Physiology Journal,2014,509:1367-1373.(in Chinese)
[26]CHONG J,PIRON M C,MEYER S,MEYER S,MERDINOGLU D,BERTSCH C,MESTRE P.The SWEET family of sugar transporters in grapevine:Vv SWEET4 is involved in the interaction with Botrytis cinerea.Journal of Experimental Botany,2014,65(22):6589-6601.
[27]赵文婷,魏建和,刘晓东,高志晖.植物瞬时表达技术的主要方法与应用进展.生物技术通讯,2013,2:294-300.ZHAO W T,WEI J H,LIU X D,GAO Z H.Advance of the main methods and applications of plant transient expression system.Letters in Biotechnology,2013,2:294-300.(in Chinese)
[28]常丽娟,刘勇,宋君,刘文娟,张富丽,王东,尹全,雷绍荣.转基因植物中外源基因的沉默及应对策略.生物技术通讯,2013,24(6):881-885.CHANG L J,LIU Y,SONG J,LIU W J,ZHANG F L,WANG D,YIN Q,LEI S R.Silence of exogenous genes in transgenic plants and the coping strategies.Letters in Biotechnology,2013,24(6):881-885.(in Chinese)
[29]YUAN M,ZHAO J,HUANG R,LI X,XIAO J,WANG S.Rice Mt N3/saliva/SWEET gene family:Evolution,expression profiling,and sugar transport.Journal of Integrative Plant Biology,2014,56(6):559-570.
[30]BüTTNER M,SAUER N.Monosaccharide transporters in plants:Structure,function and physiology.Biochimica et Biophysica Acta(BBA)-Biomembranes,2000,1465(1):263-274.
[31]LALONDE S,WIPF D,FROMMER W B.Transport mechanisms for organic forms of carbon and nitrogen between source and sink.Annual Review of Plant Biology,2004,55:341-372.