东方百合‘索邦’GA20ox的克隆及表达分析
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摘要
以东方百合‘索邦’为材料,利用cDNA末端快速扩增技术(RACE)获得编码活性GAs合成过程中双加氧酶的基因GA20ox,对其进行生物信息学分析和表达分析。结果表明,GA20ox全长1 502bp,包括1个1167bp的开放阅读框(ORF),编码388个氨基酸。GA20ox编码的蛋白存在多个糖基化位点、磷酸化位点和酰化位点。‘索邦’GA20ox和其他物种的GA20ox蛋白有很高的相似性,均含有保守的PLN02276结构域。‘索邦’GA20ox与姬蝴蝶兰、铁皮石斛、海枣、油棕和椰子等单子叶植物的GA20ox蛋白具有较高的同源性。实时荧光定量PCR分析表明,GA20ox具有时空表达特异性,在萌芽期之后的茎生根、现蕾期的茎、萌芽期和展叶期的叶片、萌芽期的顶芽等快速发育的组织中有较高的表达。在萌芽期对植株进行高浓度(200和400 mg·L~(-1))GA3处理对其生长的影响最大,其中200 mg·L~(-1) GA3处理最有利于株高的增加,400 mg·L~(-1) GA3处理最有利于地下部分干质量的增加。对萌芽期‘索邦’百合进行200 mg·L~(-1) GA3处理,显著抑制其GA20ox在除鳞片外其他部位的表达,表达量均在处理后12 h降至最低,其中在茎和基生根中降幅最大,分别下降了96.79%和94.65%。推测GA20ox的表达受活性GAs的反馈调节,在调控体内活性GAs稳态中发挥重要的作用。
We cloned a cDNA encoding GA20ox from Lilium Oriental Hybrids‘Sorbonne'using rapid amplification of cDNA ends(RACE). The bioinformatics and expression patterns of GA20ox were analyzed in our study. Sequences analysis showed that the cloned GA20ox was 1 502 bp in length with an open reading frame(1 167 bp),which encoded 388 amino acids. The putative protein had many glycosylation,phosphorylation and N-myristoylation sites. Alignment of amino acid sequence showed that GA20ox of‘Sorbonne'shared a PLN02276 binding domain like the GA20ox in other species. Phylogenetic analysis revealed that GA20ox of‘Sorbonne'was highly homologous to GA20ox of other monocots, including Phalaenopsis equestris,Dendrobium catenatum,Phoenix dactylifera,Elaeis guineensis and Cocos nucifera. Quantitative Real-time PCR(qRT-PCR)analysis showed that GA20ox was highly expressed in stem root after germination,stem of squaring stage,leaf of germination stage and leaf expansion stage,and terminal bud of germination stage,implying the temporal and special regulation of GA20ox. The application of high GA_3 concentration in germination stage had great influence on plant growth as plant height and biomass of underground were promoted by 200 mg · L~(-1) and 400 mg · L~(-1) of GA_3 treatment,respectively. After treatment of GA_3 at 200 mg · L~(-1),the expression of GA20ox was significantly suppressed in all tissues(except for bulb scale)and fell to their lowest level at 12 h,especially in the stem and root with decreasing by 96.79% and 94.65% respectively. These results suggest that the expression of GA20ox may be regulated by active gibberellins with a potential feedback mechanism,thus the GA20ox may play a key role for the maintenance of endogenetic active gibberellins homeostasis in lily.
We cloned a cDNA encoding GA20ox from Lilium Oriental Hybrids‘Sorbonne'using rapid amplification of cDNA ends(RACE). The bioinformatics and expression patterns of GA20ox were analyzed in our study. Sequences analysis showed that the cloned GA20ox was 1 502 bp in length with an open reading frame(1 167 bp),which encoded 388 amino acids. The putative protein had many glycosylation,phosphorylation and N-myristoylation sites. Alignment of amino acid sequence showed that GA20ox of‘Sorbonne'shared a PLN02276 binding domain like the GA20ox in other species. Phylogenetic analysis revealed that GA20ox of‘Sorbonne'was highly homologous to GA20ox of other monocots, including Phalaenopsis equestris,Dendrobium catenatum,Phoenix dactylifera,Elaeis guineensis and Cocos nucifera. Quantitative Real-time PCR(qRT-PCR)analysis showed that GA20ox was highly expressed in stem root after germination,stem of squaring stage,leaf of germination stage and leaf expansion stage,and terminal bud of germination stage,implying the temporal and special regulation of GA20ox. The application of high GA_3 concentration in germination stage had great influence on plant growth as plant height and biomass of underground were promoted by 200 mg · L~(-1) and 400 mg · L~(-1) of GA_3 treatment,respectively. After treatment of GA_3 at 200 mg · L~(-1),the expression of GA20ox was significantly suppressed in all tissues(except for bulb scale)and fell to their lowest level at 12 h,especially in the stem and root with decreasing by 96.79% and 94.65% respectively. These results suggest that the expression of GA20ox may be regulated by active gibberellins with a potential feedback mechanism,thus the GA20ox may play a key role for the maintenance of endogenetic active gibberellins homeostasis in lily.
引文
Carrera E,Bou J,García-Martínez J L,Prat S.2000.Changes in GA 20-oxidase gene expression strongly affect stem length,tuber induction and tuber yield of potato plants.The Plant Journal,22(3):247-256.
Choi Y H,Yoshizawa K,Kobayashi M,Sakurai A.1995.Distribution of endogenous gibberellins in vegetative shoots of rice.Plant and Cell Physiology,36:997-1001.
Coles J P,Phillips A L,Croker S J,Garcia-Lepe R,Lewis M J,Hedden P.1999.Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellin 20-oxidase genes.The Plant Journal,17(5):547-556.
Desgagné-Penix I,Sponsel V M.2008.Expression of gibberellin 20-oxidase1(AtGA20ox1)in Arabidopsis seedlings with altered auxin status is regulated at multiple levels.Journal of Experimental Botany,59(8):2057-2070.
Do P T,De Tar J R,Lee H,Folta M K,Zhang Z J.2016.Expression of ZmGA20ox cDNA alters plant morphology and increases biomass production of switchgrass(Panicum virgatum L.).Plant Biotechnology Journal,14:1532-1540.
Du J K,Yao Y Y,Ni Z F,Sun Q X.2009.Cloning and characterization of an up-regulated GA 20-oxidase gene in hybrid maize.Progress in Natural Science,19:161-166.
Eriksson M E,Israelsson M,Olsson O,Moritz T.2000.Increased gibberellin biosynthesis in transgenic trees promotes growth,biomass production and xylem fiber length.Nature Biotechnology,18:784-788.
Hedden P,Kamiya Y.1997.Gibberellin biosynthesis:enzymes,genes and their regulation.Annual Review of Plant Physiology and Plant Molecular Biology,48:431-460.
Hedden P,Phillips A L.2000.Gibberellin metabolism:New insights revealed by the genes.Trends in Plant Science,5(12):523-530.
Huang Tao-peng,Li Mei-juan,Wang Rui,Li Ling.2015.Progress in study of gibberellins biosynthesis and signaling transduction pathway.Plant Physiology Journal,51(8):1241-1247.(in Chinese)黄桃鹏,李媚娟,王睿,李玲.2015.赤霉素生物合成及信号转导途径研究进展.植物生理学报,51(8):1241-1247.
Huang S,Raman A S,Ream J E,Fujiwara H,Cerny R E,Brown S M.1998.Overexpression of 20-oxidase confers a gibberellin-overproduction phenotype in Arabidopsis.Plant Physiology,118:773-781.
Huerta L,Garcia-Lor A,Garcia-Martinez J L.2009.Characterization of gibberellin 20-oxidases in the citrus hybrid Carrizocit range.Tree Physiology,29:569-577.
Ji Lan,Yang Ren-cui.2002.Rice stem elongation and plant hormones.Chinese Bulletin of Botany,19(1):109-115.(in Chinese)季兰,杨仁崔.2002.水稻茎伸长生长与植物激素.植物学通报,19(1):109-115.
Jiang Zhi-ang,Sun Jian-she,Peng Jian-ying,Shao Jian-zhu.2013.Isolation,subcellular location and expression of MdGA20ox1 gene from apple.Acta Horticulturae Sinica,40(12):2373-2381.(in Chinese)姜志昂,孙建设,彭建营,邵建柱.2013.苹果MdGA20ox1基因的克隆、亚细胞定位及表达分析.园艺学报,40(12):2373-2381.
Kamiya Y,García-Mart?ez J L.1999.Regulation of gibberellin biosynthesis by light.Current Opinion in Plant Biology,2:398-403.
Kang H G,Jun S H,Kim J,Kawaide H,Kamiya Y,An G.1999.Cloning and molecular analyses of a gibberellin 20-oxidase gene expressed specifically in developing seeds of watermelon.Plant Physiology,121:373-382.
Li Wei,Chen Xiao-yang,Ding Xia.2003.Studies on the GA20-oxidase of higher plants.Chinese Bulletin of Life Sciences,15(1):42-45.(in Chinese)李伟,陈晓阳,丁霞.2003.高等植物GA20-氧化酶研究进展.生命科学,15(1):42-45.
Li Yan-bing,Zhu Mei-yu,Yang Xiao-ru,Li Yue,He Yi,Qi Ming-fang,Li Tian-lai.2018.Advances on Gibberellin 2-oxidases gene in higher plants.Acta Horticulturae Sinica,45(9):1844-1856.(in Chinese)李艳冰,朱美玉,杨晓茹,李悦,赫易,齐明芳,李天来.2018.植物中Gibberellin 2-oxidases基因研究进展.园艺学报,45(9):1844-1856.
Liu Wen-chao,Wang Dong-hao,Wang Zhe-zhi,Li Cui-qin.2012.Cloning and expression analysis of 2-oxoglutarate-dependent dioxygenase from Salvia miltiorrhiza Bunge.Acta Botanica Boreali-Occidentalia Sinica,32(7):1289-1294.(in Chinese)刘文超,王东浩,王喆之,李翠芹.2012.丹参2-酮戊二酸依赖性双加氧酶基因克隆及表达分析.西北植物学报,32(7):1289-1294.
Mutasa-G?ttgens E,Hedden P.2009.Gibberellin as a factor in floral regulatory networks.Journal of Experimental Botany,60(7):1979-1989.
Olszewski N,Sun T P,Gubler F.2002.Gibberellin Signaling:biosynthesis,catabolism,and response pathways.The Plant Cell,14:S61-S80.
Phillips A L,Ward D A,Uknes S,Appleford N E,Lange T,Huttly A K,Gaskin P,Craebe J E,Hedden P.1995.Isolation and expression of three gibberellin 20-oxidase cDNA clones from Arabidopsis.Plant Physiology,108:1049-1057.
Pimenta Lange M J,Lange T.2006.Gibberellin biosynthesis and the regulation of plant development.Plant Biology,(8):281-290.
Qiao Feng,Wang Chun-lian,Chen Zhi,Geng Gui-gong.2013.Influence on plant architecture through the over expression of OsGA20ox2 in rice.Journal of China Agricultural University,18(1):25-31.(in Chinese)乔枫,王春连,陈志,耿贵工.2013.OsGA20ox2基因过量表达对水稻性状的影响.中国农业大学学报,18(1):25-31.
Richards D E,King K E,Ait-Ali T,Hardberd N P.2001.How gibberellin regulates plant growth and development:a molecular genetic analysis of gibberellin signaling.Annual Review of Plant Physiology and Plant Molecular Biology,52:67-88.
Seymour G B,?stergaard L,Chapman N H,Knapp S,Martin C.2013.Fruit development and ripening.Annual Review of Plant Biology,64:219-241.
Toyomasu T,Kawaide H,Sekimoto H,Numers C V,Phillips A L,Hedden P,Kamiya Y.1997.Cloning and characterization of a cDNA encoding gibberellin 20-oxidase from rice(Oryza sativa)seedlings.Physiologia Plantarum,99:111-118.
Vidal A M,Ben-Cheikh W,Talon M,García-Martínez J L.2003.Regulation of gibberellin 20-oxidase gene expression and gibberellin content in citrus by temperature and citrus exocortis viroid.Planta,217(3):442-448.
Wang Jiang-ying,Wu Bin,Liu Wei-xin,Fan Zheng-qi,Li Ji-yuan.2015.Overexpression and silence expression of CrGA20ox1 from Camellia reticulata‘Hentiangao’and its effect on plant forms in transgenic tobacco.Acta Horticulturae Sinica,42(8):1523-1532.(in Chinese)王江英,吴斌,刘伟鑫,范正琪,李纪元.2015.矮生山茶‘恨天高’GA20ox1的克隆及其对转基因烟草株形的影响.园艺学报,42(8):1523-1532.
Wolbang C M,Chandler P M,Smith J J,Ross J J.2004.Auxin from the developing inflorescence is required for the biosynthesis of active gibberellins in barley stems.Plant Physiology,134(2):769-776.
Wu Jian-ming,Li Yang-rui,Yang Liu,Wang Ai-qin,Yang Li-tao.2009.Relationship between gibberellin-induced internode elongation and endogenous hormone changes in sugarcan.Chinese Journal of Tropical Crops,30(10):1452-1457.(in Chinese)吴建明,李杨瑞,杨柳,王爱勤,杨丽涛.2009.赤霉素诱导甘蔗节间伸长与内源激素变化的关系.热带作物学报,30(10):1452-1457.
Xu Y L,Li L,Wu K,Peeters A J,Gage D A,Zeevaart J A.1995.The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase:molecular cloning and functional expression.Proceedings of the National Academy of Sciences of the United States of America,92:6640-6644.
Yamaguchi S,Kamiya Y.2000.Gibberellin biosynthesis:its regulation by endogenous and environmental signals.Plant and Cell Physiology,41:251-257.
Yamaguchi S.2008.Gibberellin metabolism and its regulation.Annual Review of Plant Biology,59:225-251.
Choi Y H,Yoshizawa K,Kobayashi M,Sakurai A.1995.Distribution of endogenous gibberellins in vegetative shoots of rice.Plant and Cell Physiology,36:997-1001.
Coles J P,Phillips A L,Croker S J,Garcia-Lepe R,Lewis M J,Hedden P.1999.Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellin 20-oxidase genes.The Plant Journal,17(5):547-556.
Desgagné-Penix I,Sponsel V M.2008.Expression of gibberellin 20-oxidase1(AtGA20ox1)in Arabidopsis seedlings with altered auxin status is regulated at multiple levels.Journal of Experimental Botany,59(8):2057-2070.
Do P T,De Tar J R,Lee H,Folta M K,Zhang Z J.2016.Expression of ZmGA20ox cDNA alters plant morphology and increases biomass production of switchgrass(Panicum virgatum L.).Plant Biotechnology Journal,14:1532-1540.
Du J K,Yao Y Y,Ni Z F,Sun Q X.2009.Cloning and characterization of an up-regulated GA 20-oxidase gene in hybrid maize.Progress in Natural Science,19:161-166.
Eriksson M E,Israelsson M,Olsson O,Moritz T.2000.Increased gibberellin biosynthesis in transgenic trees promotes growth,biomass production and xylem fiber length.Nature Biotechnology,18:784-788.
Hedden P,Kamiya Y.1997.Gibberellin biosynthesis:enzymes,genes and their regulation.Annual Review of Plant Physiology and Plant Molecular Biology,48:431-460.
Hedden P,Phillips A L.2000.Gibberellin metabolism:New insights revealed by the genes.Trends in Plant Science,5(12):523-530.
Huang Tao-peng,Li Mei-juan,Wang Rui,Li Ling.2015.Progress in study of gibberellins biosynthesis and signaling transduction pathway.Plant Physiology Journal,51(8):1241-1247.(in Chinese)黄桃鹏,李媚娟,王睿,李玲.2015.赤霉素生物合成及信号转导途径研究进展.植物生理学报,51(8):1241-1247.
Huang S,Raman A S,Ream J E,Fujiwara H,Cerny R E,Brown S M.1998.Overexpression of 20-oxidase confers a gibberellin-overproduction phenotype in Arabidopsis.Plant Physiology,118:773-781.
Huerta L,Garcia-Lor A,Garcia-Martinez J L.2009.Characterization of gibberellin 20-oxidases in the citrus hybrid Carrizocit range.Tree Physiology,29:569-577.
Ji Lan,Yang Ren-cui.2002.Rice stem elongation and plant hormones.Chinese Bulletin of Botany,19(1):109-115.(in Chinese)季兰,杨仁崔.2002.水稻茎伸长生长与植物激素.植物学通报,19(1):109-115.
Jiang Zhi-ang,Sun Jian-she,Peng Jian-ying,Shao Jian-zhu.2013.Isolation,subcellular location and expression of MdGA20ox1 gene from apple.Acta Horticulturae Sinica,40(12):2373-2381.(in Chinese)姜志昂,孙建设,彭建营,邵建柱.2013.苹果MdGA20ox1基因的克隆、亚细胞定位及表达分析.园艺学报,40(12):2373-2381.
Kamiya Y,García-Mart?ez J L.1999.Regulation of gibberellin biosynthesis by light.Current Opinion in Plant Biology,2:398-403.
Kang H G,Jun S H,Kim J,Kawaide H,Kamiya Y,An G.1999.Cloning and molecular analyses of a gibberellin 20-oxidase gene expressed specifically in developing seeds of watermelon.Plant Physiology,121:373-382.
Li Wei,Chen Xiao-yang,Ding Xia.2003.Studies on the GA20-oxidase of higher plants.Chinese Bulletin of Life Sciences,15(1):42-45.(in Chinese)李伟,陈晓阳,丁霞.2003.高等植物GA20-氧化酶研究进展.生命科学,15(1):42-45.
Li Yan-bing,Zhu Mei-yu,Yang Xiao-ru,Li Yue,He Yi,Qi Ming-fang,Li Tian-lai.2018.Advances on Gibberellin 2-oxidases gene in higher plants.Acta Horticulturae Sinica,45(9):1844-1856.(in Chinese)李艳冰,朱美玉,杨晓茹,李悦,赫易,齐明芳,李天来.2018.植物中Gibberellin 2-oxidases基因研究进展.园艺学报,45(9):1844-1856.
Liu Wen-chao,Wang Dong-hao,Wang Zhe-zhi,Li Cui-qin.2012.Cloning and expression analysis of 2-oxoglutarate-dependent dioxygenase from Salvia miltiorrhiza Bunge.Acta Botanica Boreali-Occidentalia Sinica,32(7):1289-1294.(in Chinese)刘文超,王东浩,王喆之,李翠芹.2012.丹参2-酮戊二酸依赖性双加氧酶基因克隆及表达分析.西北植物学报,32(7):1289-1294.
Mutasa-G?ttgens E,Hedden P.2009.Gibberellin as a factor in floral regulatory networks.Journal of Experimental Botany,60(7):1979-1989.
Olszewski N,Sun T P,Gubler F.2002.Gibberellin Signaling:biosynthesis,catabolism,and response pathways.The Plant Cell,14:S61-S80.
Phillips A L,Ward D A,Uknes S,Appleford N E,Lange T,Huttly A K,Gaskin P,Craebe J E,Hedden P.1995.Isolation and expression of three gibberellin 20-oxidase cDNA clones from Arabidopsis.Plant Physiology,108:1049-1057.
Pimenta Lange M J,Lange T.2006.Gibberellin biosynthesis and the regulation of plant development.Plant Biology,(8):281-290.
Qiao Feng,Wang Chun-lian,Chen Zhi,Geng Gui-gong.2013.Influence on plant architecture through the over expression of OsGA20ox2 in rice.Journal of China Agricultural University,18(1):25-31.(in Chinese)乔枫,王春连,陈志,耿贵工.2013.OsGA20ox2基因过量表达对水稻性状的影响.中国农业大学学报,18(1):25-31.
Richards D E,King K E,Ait-Ali T,Hardberd N P.2001.How gibberellin regulates plant growth and development:a molecular genetic analysis of gibberellin signaling.Annual Review of Plant Physiology and Plant Molecular Biology,52:67-88.
Seymour G B,?stergaard L,Chapman N H,Knapp S,Martin C.2013.Fruit development and ripening.Annual Review of Plant Biology,64:219-241.
Toyomasu T,Kawaide H,Sekimoto H,Numers C V,Phillips A L,Hedden P,Kamiya Y.1997.Cloning and characterization of a cDNA encoding gibberellin 20-oxidase from rice(Oryza sativa)seedlings.Physiologia Plantarum,99:111-118.
Vidal A M,Ben-Cheikh W,Talon M,García-Martínez J L.2003.Regulation of gibberellin 20-oxidase gene expression and gibberellin content in citrus by temperature and citrus exocortis viroid.Planta,217(3):442-448.
Wang Jiang-ying,Wu Bin,Liu Wei-xin,Fan Zheng-qi,Li Ji-yuan.2015.Overexpression and silence expression of CrGA20ox1 from Camellia reticulata‘Hentiangao’and its effect on plant forms in transgenic tobacco.Acta Horticulturae Sinica,42(8):1523-1532.(in Chinese)王江英,吴斌,刘伟鑫,范正琪,李纪元.2015.矮生山茶‘恨天高’GA20ox1的克隆及其对转基因烟草株形的影响.园艺学报,42(8):1523-1532.
Wolbang C M,Chandler P M,Smith J J,Ross J J.2004.Auxin from the developing inflorescence is required for the biosynthesis of active gibberellins in barley stems.Plant Physiology,134(2):769-776.
Wu Jian-ming,Li Yang-rui,Yang Liu,Wang Ai-qin,Yang Li-tao.2009.Relationship between gibberellin-induced internode elongation and endogenous hormone changes in sugarcan.Chinese Journal of Tropical Crops,30(10):1452-1457.(in Chinese)吴建明,李杨瑞,杨柳,王爱勤,杨丽涛.2009.赤霉素诱导甘蔗节间伸长与内源激素变化的关系.热带作物学报,30(10):1452-1457.
Xu Y L,Li L,Wu K,Peeters A J,Gage D A,Zeevaart J A.1995.The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase:molecular cloning and functional expression.Proceedings of the National Academy of Sciences of the United States of America,92:6640-6644.
Yamaguchi S,Kamiya Y.2000.Gibberellin biosynthesis:its regulation by endogenous and environmental signals.Plant and Cell Physiology,41:251-257.
Yamaguchi S.2008.Gibberellin metabolism and its regulation.Annual Review of Plant Biology,59:225-251.