苹果糖转运蛋白基因MdSWEET17的功能鉴定
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摘要
苹果糖转运蛋白基因MdSWEET17在液泡膜糖转运过程中发挥着重要的作用。本文以新疆红肉苹果杂种一代优系‘红脆1号’为试材,克隆Md SWEET17并原核诱导获得其重组蛋白,对其进行生物信息学分析;测定该基因在不同组织及不同发育时期的表达,通过亚细胞定位及转基因鉴定其功能。结果显示, Md SWEET17基因编码区长度为786 bp,蛋白大小为29.34 kDa左右,定位于15号染色体,由5个外显子和4个内含子组成。糖代谢相关转运蛋白系统进化树分析发现MdSWEET17与AtSWEET17在同一个进化树枝上; MdSWEET17定位于苹果‘王林’愈伤组织原生质体的液泡膜上。定量表达分析表明, Md SWEET17在苹果的根、叶和幼果中均有较高的表达,且在果实发育中, Md SWEET17的表达水平与果糖含量显著负相关。Md SWEET17启动子含有与糖信号及激素信号相关的顺式作用元件。低温和糖处理‘王林’愈伤组织后发现Md SWEET17响应果糖和低温诱导;而在‘王林’愈伤组织中过表达Md SWEET17,发现它对葡萄糖和蔗糖含量没有影响,但能减少‘王林’愈伤组织的果糖含量。
The apple sugar transporter gene MdSWEET17 played an important role in tonoplast sugar transport. We cloned the MdSWEET17 in apple 'Hongcui No.1' and obtained the recombinant protein by the prokaryotic induction technology, then, analysed the bio-information of it. The expression levels of MdWEET17 in different tissues and different development stages were studied by the qRT-PCR, and we also identificated the fuction of MdWEET17 by the subcellular localization and transgenosis. The results showed that the length of coding sequence was 786 bp and the molecular mass of MdSWEET17 was about 29.34 kDa, it was located in the chromosome 15 of the apple genome, included 5 exons and 4 introns. A phylogenetic tree of related transporter in sugar metabolism indicated MdSWEET17 and AtSWEET17 were located in the same evolutionary branch. MdSWEET17 was located in the tonoplast of orin callus. The qRT-PCR results showed that the MdSWEET17 had the higher expression level in roots, leaves and young fruits of apples, and its expression had a significant negative correlation with the content of fructose during the development stage of fruits. The promoter of Md SWEET17 contained several typical cis-acting elements, including sugar signaling responsive elements and phytohormone responsive elements. After low temperature and sugar treatment of apple 'Orin' callus MdSWEET17 was found in response to fructose and low temperature induction. When overexpressing Md SWEET17 in the orin callus, We found it could reduce the contents of fructose, however, it had no influence on the contents of sucrose and glucose.
The apple sugar transporter gene MdSWEET17 played an important role in tonoplast sugar transport. We cloned the MdSWEET17 in apple 'Hongcui No.1' and obtained the recombinant protein by the prokaryotic induction technology, then, analysed the bio-information of it. The expression levels of MdWEET17 in different tissues and different development stages were studied by the qRT-PCR, and we also identificated the fuction of MdWEET17 by the subcellular localization and transgenosis. The results showed that the length of coding sequence was 786 bp and the molecular mass of MdSWEET17 was about 29.34 kDa, it was located in the chromosome 15 of the apple genome, included 5 exons and 4 introns. A phylogenetic tree of related transporter in sugar metabolism indicated MdSWEET17 and AtSWEET17 were located in the same evolutionary branch. MdSWEET17 was located in the tonoplast of orin callus. The qRT-PCR results showed that the MdSWEET17 had the higher expression level in roots, leaves and young fruits of apples, and its expression had a significant negative correlation with the content of fructose during the development stage of fruits. The promoter of Md SWEET17 contained several typical cis-acting elements, including sugar signaling responsive elements and phytohormone responsive elements. After low temperature and sugar treatment of apple 'Orin' callus MdSWEET17 was found in response to fructose and low temperature induction. When overexpressing Md SWEET17 in the orin callus, We found it could reduce the contents of fructose, however, it had no influence on the contents of sucrose and glucose.
引文
Agarwal M,Hao Y,Kapoor A,et al(2006).A R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance.JBiol Chem,281(49):37636-37645
Bush DR(1993).Proton-coupled sugar and amino acid transporters in plants.Annu Rev Plant Physiol,44(1):513-542
Chardon F,Bedu M,Calenge F,et al(2013).Leaf fructose content is controlled by the vacuolar transporter SWEET17 in Arabidopsis.Curr Biol,23:697-702
Chen LQ,Hou BH,Lalonde S,et al(2010).Sugar transporters for intercellular exchange and nutrition of pathogens.Nature,468(7323):527-532
Chen LQ,Qu XQ,Hou BH,et al(2012).Sucrose efflux mediated by SWEET proteins as a key step for phloem transport.Science,335:207-211
Chen XS,Feng T,Zhang YM,et al(2007).Genetic diversity of volatile components in Xinjiang wild apple(Malus sieversii).J Genet Genomics,34(2):171-179
Fusada N,Masuda T,Kuroda H,et al(2005).Identification of a novel cis-element exhibiting cytokinin-dependent protein binding in vitro in the 5′-region of NADPH-protochlorophyllide oxidoreductase gene in cucumber.Plant Mol Biol,59(4):631-645
Geiger D(2011).Plant sucrose transporters from a biophysical point of view.Mol Plant,4:395-406
Ji XH,Wang YT,Zhang R,et al(2015).Effect of auxin,cytokinin and nitrogen on anthocyanin biosynthesis in callus cultures of red-fleshed apple(Malus sieversii,f.niedzwetzkyana).Plant Cell Tiss Org Cult Cult,120(1):325-337
Katz E,Fon M,Lee YJ,et al(2007).The citrus fruit proteome:insights into citrus fruit metabolism.Planta,226:989-1005
Klemens PA,Patzke K,Trentmann O,et al(2014).Overexpression of a proton-coupled vacuolar glucose exporter impairs freezing tolerance and seed germination.New Phytol,202:188-197
Lin IW,Sosso D,Chen L Q,et al(2014).Nectar secretion requires sucrose phosphate synthases and the sugar transporter SWEET9.Nature,508:546-549
Livak KJ,Schmittgen TD(2001).Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method.Methods,25(4):402-408
Martinoia E,Meyer S,De Angeli A,et al(2012).Vacuolar transporters in their physiological context.Annu Rev Plant Biol,63:183-213
Rae A,Perroux JM,Grof CPL(2005).Sucrose partitioning between vascular bundles and storage parenchyma in the sugarcane stem:apotential role for the ShSUT1 sucrose transporter.Planta,220(6):817-825
Ruan YL(2014).Sucrose metabolism:gateway to diverse carbon use and sugar signaling.Annu Rev Plant Biol,65:33-67
Schneider S,Hulpke S,Schulz A,et al(2012).Vacuoles release sucrose via tonoplast-localised SUC4-type transporters.Plant Biol,14:325-336
Schulz A,Beyhl D,Marten I,et al(2011).Proton-driven sucrose symport and antiport is provided by the vacuolar transporters SUC4 and TMT1/2.Plant J,68:129-136
Simpson SD,Nakashima K,Narusaka Y,et al(2003).Two different novel cis-acting elements of erd1,a clpA homologous Arabidopsis gene function in induction by dehydration stress and dark-induced senescence.Plant J,33(2):259-270
Solano R,Nieto C,Avila J,et al(1995).Dual DNA binding specificity of a petal epidermis-specific MYB transcription factor(MYB.Ph3)from Petunia hybrida.EMBO J,14(8):1773-1784
Tatematsu K,Ward S,Leyser O,et al(2005).Identification of cis-elements that regulate gene expression during initiation of axillary bud ougtgrowth in Arabidopsis.Plant Physiol,138(2):757-766
Urao T,Yamaguchi-Shinozaki K,Urao S,et al(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
Wingenter K,Schulz A,Wormit A,et al(2010).Increased activity of the vacuolar monosaccharide transporter TMT1alters cellular sugar partitioning,sugar signaling,and seed yield in Arabidopsis.Plant Physiol,154(2):665-677
Will A,Tanner W(1996).Importance of the first external loop for substrate recognition as revealed by chimeric Chlorella monosaccharide/H symporters.FEBS Lett,381:127-130
Xu HF,Liu JX,Wang YC,et al(2016).Isolation and expression analysis of a vacuolar glucose transporter gene MdVGT1 in apple.Sci Agric Sin,49(23):4584-4592(in Chinese with English abstract)[许海峰,刘静轩,王意程等(2016).苹果液泡膜葡萄糖转运蛋白基因MdVGT1的克隆与表达分析.中国农业科学,49(23):4584-4592]
Zhao ZZ,Zhang SL,Xu CJ,et al(2001).Roles of activities of sucrose-metabolizing enzymes in accumulation of sugars in satsuma mandarin fruit.Acta Hortic Sin,28(2):112-118(in Chinese with English abstract)[赵智中,张上隆,徐昌杰等(2001).蔗糖代谢相关酶在温州蜜柑果实糖积累中的作用.园艺学报,28(2):112-118
Zhou Y,Grof CPL,Patrick JW(2014).Proof of concept for a novel functional screening system for plant sucrose effluxers.J Biol Methods,1(2):e5.1-e5.6
Bush DR(1993).Proton-coupled sugar and amino acid transporters in plants.Annu Rev Plant Physiol,44(1):513-542
Chardon F,Bedu M,Calenge F,et al(2013).Leaf fructose content is controlled by the vacuolar transporter SWEET17 in Arabidopsis.Curr Biol,23:697-702
Chen LQ,Hou BH,Lalonde S,et al(2010).Sugar transporters for intercellular exchange and nutrition of pathogens.Nature,468(7323):527-532
Chen LQ,Qu XQ,Hou BH,et al(2012).Sucrose efflux mediated by SWEET proteins as a key step for phloem transport.Science,335:207-211
Chen XS,Feng T,Zhang YM,et al(2007).Genetic diversity of volatile components in Xinjiang wild apple(Malus sieversii).J Genet Genomics,34(2):171-179
Fusada N,Masuda T,Kuroda H,et al(2005).Identification of a novel cis-element exhibiting cytokinin-dependent protein binding in vitro in the 5′-region of NADPH-protochlorophyllide oxidoreductase gene in cucumber.Plant Mol Biol,59(4):631-645
Geiger D(2011).Plant sucrose transporters from a biophysical point of view.Mol Plant,4:395-406
Ji XH,Wang YT,Zhang R,et al(2015).Effect of auxin,cytokinin and nitrogen on anthocyanin biosynthesis in callus cultures of red-fleshed apple(Malus sieversii,f.niedzwetzkyana).Plant Cell Tiss Org Cult Cult,120(1):325-337
Katz E,Fon M,Lee YJ,et al(2007).The citrus fruit proteome:insights into citrus fruit metabolism.Planta,226:989-1005
Klemens PA,Patzke K,Trentmann O,et al(2014).Overexpression of a proton-coupled vacuolar glucose exporter impairs freezing tolerance and seed germination.New Phytol,202:188-197
Lin IW,Sosso D,Chen L Q,et al(2014).Nectar secretion requires sucrose phosphate synthases and the sugar transporter SWEET9.Nature,508:546-549
Livak KJ,Schmittgen TD(2001).Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method.Methods,25(4):402-408
Martinoia E,Meyer S,De Angeli A,et al(2012).Vacuolar transporters in their physiological context.Annu Rev Plant Biol,63:183-213
Rae A,Perroux JM,Grof CPL(2005).Sucrose partitioning between vascular bundles and storage parenchyma in the sugarcane stem:apotential role for the ShSUT1 sucrose transporter.Planta,220(6):817-825
Ruan YL(2014).Sucrose metabolism:gateway to diverse carbon use and sugar signaling.Annu Rev Plant Biol,65:33-67
Schneider S,Hulpke S,Schulz A,et al(2012).Vacuoles release sucrose via tonoplast-localised SUC4-type transporters.Plant Biol,14:325-336
Schulz A,Beyhl D,Marten I,et al(2011).Proton-driven sucrose symport and antiport is provided by the vacuolar transporters SUC4 and TMT1/2.Plant J,68:129-136
Simpson SD,Nakashima K,Narusaka Y,et al(2003).Two different novel cis-acting elements of erd1,a clpA homologous Arabidopsis gene function in induction by dehydration stress and dark-induced senescence.Plant J,33(2):259-270
Solano R,Nieto C,Avila J,et al(1995).Dual DNA binding specificity of a petal epidermis-specific MYB transcription factor(MYB.Ph3)from Petunia hybrida.EMBO J,14(8):1773-1784
Tatematsu K,Ward S,Leyser O,et al(2005).Identification of cis-elements that regulate gene expression during initiation of axillary bud ougtgrowth in Arabidopsis.Plant Physiol,138(2):757-766
Urao T,Yamaguchi-Shinozaki K,Urao S,et al(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
Wingenter K,Schulz A,Wormit A,et al(2010).Increased activity of the vacuolar monosaccharide transporter TMT1alters cellular sugar partitioning,sugar signaling,and seed yield in Arabidopsis.Plant Physiol,154(2):665-677
Will A,Tanner W(1996).Importance of the first external loop for substrate recognition as revealed by chimeric Chlorella monosaccharide/H symporters.FEBS Lett,381:127-130
Xu HF,Liu JX,Wang YC,et al(2016).Isolation and expression analysis of a vacuolar glucose transporter gene MdVGT1 in apple.Sci Agric Sin,49(23):4584-4592(in Chinese with English abstract)[许海峰,刘静轩,王意程等(2016).苹果液泡膜葡萄糖转运蛋白基因MdVGT1的克隆与表达分析.中国农业科学,49(23):4584-4592]
Zhao ZZ,Zhang SL,Xu CJ,et al(2001).Roles of activities of sucrose-metabolizing enzymes in accumulation of sugars in satsuma mandarin fruit.Acta Hortic Sin,28(2):112-118(in Chinese with English abstract)[赵智中,张上隆,徐昌杰等(2001).蔗糖代谢相关酶在温州蜜柑果实糖积累中的作用.园艺学报,28(2):112-118
Zhou Y,Grof CPL,Patrick JW(2014).Proof of concept for a novel functional screening system for plant sucrose effluxers.J Biol Methods,1(2):e5.1-e5.6