牡丹休眠相关基因PsGRASs筛选、克隆和表达特性分析
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摘要
通过筛选、克隆牡丹PsGRASs基因并进行表达模式分析,初步解析其是否参与牡丹休眠解除,以期为牡丹反季节催花提供候选基因。利用HMM(Hidden Markov Model)模型,基于GRAS基因家族的种子(Pfam号为PF03514)序列,利用BioEdit软件对牡丹花芽休眠的454测序转录组数据进行本地检索,筛选到2个具有GRAS结构域的序列。通过RACE扩增得到了其全长cDNA,其中PsGRAS1序列为2 308 bp,开放阅读框(open reading frame,ORF)为1 848 bp,编码615个氨基酸;PsGRAS2为2 034bp,ORF为1560bp,编码518个氨基酸,两个编码PsGRASs蛋白的同源性为19.87%。进化树结果表明,PsGRAS1蛋白与拟南芥GRAS家族中的DELLA亚家族的GAI和RGLs聚到一个大的分支,PsGRAS2蛋白与拟南芥HAMs(AtSCL6/15/22/26/27)并为一支,说明PsGRAS1和PsGRAS2来自GRAS家族的两个不同分支。组织表达结果显示PsGRAS1和PsGRAS2在牡丹初花期不同组织中表达水平不同,PsGRAS1在叶中表达水平最高,在雄蕊中最低;而PsGRAS2在苞片中表达水平最高,在花瓣中最低。在牡丹花芽休眠解除过程中,PsGRAS1呈下调趋势,低温28 d表达水平最低,而PsGRAS2呈先上调后下调趋势,低温14 d表达水平最高。在外源GA3处理7 d的牡丹花芽中,PsGRAS1的表达显著下降,而PsGRAS2显著提高。这表明PsGRAS1和PsGRAS2都参与了休眠过程,但功能不同。
By screening and cloning the PsGRASs gene of peony and analyzing its expression pattern,the author preliminarily analyzed whether it participated in the dormancy release of peony,in order to provide candidate genes for anti-seasonal flower urge of peony. Based on the seed sequence of the GRAS gene family(Pfam No. PF03514),local blast was run to search GRAS gene family members in 454 sequencing transcriptomic database using the Hidden Markov Model(HMM)model and BioEdit software.As a result,two ESTs with GRAS domain were found. The full-length cDNAs were obtained by RACE.The full cDNA sequence of PsGRAS1 was 2 308 bp,the open reading frame(ORF)was 1 848 bp,encoding615 amino acids;the PsGRAS2 was 2 034 bp,and the ORF was 1 560 bp,encoding 518 amino acids. The homology of the two PsGRAS proteins was 19.87%. The results of phylogenetic tree showed that PsGRAS1 protein and GA1,RGLs of DELLA subfamily in Arabidopsis were clustered into a large branch,PsGRAS2 protein is in a branch with Arabidopsis HAMs(AtSCL6/15/22/26/27),indicating that two GRAS members are from two different branches of the GRAS family. The tissue expression results showed that PsGRAS1 and PsGRAS2 were expressed in different tissues at the early stage of peony flowering. The expression level of PsGRAS1 was the highest in leaves and the lowest in stamens,but that of PsGRAS2 in bracts was the highest,and the lowest in the petals. PsGRAS1 genes showed a down-regulation pattern during the dormancy release,and had the lowest expression level after 28 d chilling treatment,while PsGRAS2 showed an up-regulation trend with the highest transcript level after 14 d chilling treatment and followed by a downward trend. After being treated with exogenous GA3 for 7 days,the PsGRAS1 gene showed a dramatically decreasing trend and the PsGRAS2 gene was significantly increased. These results indicated that both PsGRAS1 and PsGRAS2 were involved in the dormancy process with different functions.
By screening and cloning the PsGRASs gene of peony and analyzing its expression pattern,the author preliminarily analyzed whether it participated in the dormancy release of peony,in order to provide candidate genes for anti-seasonal flower urge of peony. Based on the seed sequence of the GRAS gene family(Pfam No. PF03514),local blast was run to search GRAS gene family members in 454 sequencing transcriptomic database using the Hidden Markov Model(HMM)model and BioEdit software.As a result,two ESTs with GRAS domain were found. The full-length cDNAs were obtained by RACE.The full cDNA sequence of PsGRAS1 was 2 308 bp,the open reading frame(ORF)was 1 848 bp,encoding615 amino acids;the PsGRAS2 was 2 034 bp,and the ORF was 1 560 bp,encoding 518 amino acids. The homology of the two PsGRAS proteins was 19.87%. The results of phylogenetic tree showed that PsGRAS1 protein and GA1,RGLs of DELLA subfamily in Arabidopsis were clustered into a large branch,PsGRAS2 protein is in a branch with Arabidopsis HAMs(AtSCL6/15/22/26/27),indicating that two GRAS members are from two different branches of the GRAS family. The tissue expression results showed that PsGRAS1 and PsGRAS2 were expressed in different tissues at the early stage of peony flowering. The expression level of PsGRAS1 was the highest in leaves and the lowest in stamens,but that of PsGRAS2 in bracts was the highest,and the lowest in the petals. PsGRAS1 genes showed a down-regulation pattern during the dormancy release,and had the lowest expression level after 28 d chilling treatment,while PsGRAS2 showed an up-regulation trend with the highest transcript level after 14 d chilling treatment and followed by a downward trend. After being treated with exogenous GA3 for 7 days,the PsGRAS1 gene showed a dramatically decreasing trend and the PsGRAS2 gene was significantly increased. These results indicated that both PsGRAS1 and PsGRAS2 were involved in the dormancy process with different functions.
引文
Bolle C.2004.The role of GRAS proteins in plant signal transduction and development.Planta,218(5):683-692.
Duan Chengguo,Li Xianli,Gao Dongsheng,Liu Huanfang,Li Meng.2004.Studies on regulations of endogenous ABA and GA3 in sweet cherry flower buds on dormancy.Acta Horticulturae Sinica,31(2):149-154.(in Chinese)段成国,李宪利,高东升,刘焕芳,李萌.2004.内源ABA和GA3对欧洲甜樱桃花芽自然休眠的调控.园艺学报,31(2):149-154.
Finn R D,Bateman A,Clements J,Coggill P,Eberhardt R Y,Eddy S R,Heger A,Hetherington K,Holm L,Mistry J,Sonnhammer E L L,Tate J,Punta M.2014.Pfam:the protein families database.Nucleic Acids Research,42:222-230.
Gai Shupeng,Zhang Feng,Zhang Yuxi,Zheng Guosheng.2012.Analysis of genomic DNA methylation during chilling induced endo-dormancy release by methylation sensitive amplified polymorphism(MSAP)technology in tree peony(Paeonia suffruticosa).Journal of Agricultural Biotechnology,20(3):261-267.(in Chinese)盖树鹏,张风,张玉喜,郑国生.2012.低温解除牡丹休眠进程中基因组DNA甲基化敏感扩增多态性(MSAP)分析.农业生物技术学报,20(3):261-267.
Gai S P,Zhang Y X,Liu C Y,Zhang Y,Zheng G S.2013.Transcript profiling of Paoenia ostii during artificial chilling induced dormancy release identifies activation of GA pathway and carbohydrate metabolism.PLoS ONE,8(2):e55297.
Gai S P,Zhang Y X,Mu P,Liu C Y,Liu S,Dong L,Zheng G S.2012.Transcriptome analysis of tree peony during chilling requirement fulfillment:assembling,annotation and markers discovering.Gene,497(2):256-262.
Huang X,Zhu W,Dai S L,Gai S P,Zheng G S,Zheng C C.2008.The involvement of mitochondrial phosphate transporter in accelerating bud dormancy release during chilling treatment of tree peony(Paeonia suffruticosa).Planta,228(4):545-552.
Koizumi K,Gallagher K L.2013.Identification of SHRUBBY,a SHORT-ROOT and SCARECROW interacting protein that controls root growth and radial patterning.Development,140(6):1292-1300.
Li W Q,Wu J G,Weng S L,Zhang Y J,Zhang D P,Shi C H.2010.Identification and characterization of dwarf 62,a loss-of-function mutation in DLT/OsGRAS-32 affecting gibberellin metabolism in rice.Planta,232(6):1383-1396.
Livak J,Schmittgen D.2001.Analysis of relative gene expression data using real time quantitative PCR and the 2-??Ct method.Methods,25(4):402-408.
Locascio A,Blázquez M A,AlabadíD.2013.Genomic analysis of DELLA protein activity.Plant&Cell Physiology,54(8):1229-1237.
Lu Jiu-xing.2015.Functional analysis of DELLA genes related to dormancy in Prunus mume[Ph.D.Dissertation].Beijing:Beijing Forestry University.(in Chinese)逯久幸.2015.梅花休眠相关DELLA基因的克隆与功能分析[博士论文].北京:北京林业大学.
Mornya P M P,Cheng F Y.2018.Effect of combined chilling and GA3 treatment on bud abortion in forced‘Luoyanghong’tree peony(Paeonia suffruticosa Andr.).Horticultural Plant Journal,4(6):250-256.
Morohashi K,Minami M,Takase H,Hotta Y,Hiratsuka K.2003.Isolation and characterization of a novel GRAS gene that regulates meiosis-associated gene expression.Journal of Biological Chemistry,278(23):20865-20873.
Pysh L D,Wysocka-Diller J W,Camilleri C,Benfey P N.1999.The GRAS gene family in Arabidopsis:sequence characterization and basic expression analysis of the SCARECROW-LIKE genes.Plant Journal for Cell&Molecular Biology,18(1):111-119.
Song Li-li,Tao Lei,Cui Hui-ping,Ling Lei,Guo Chang-hong.2017.Genome-wide identification and expression analasis of th-e GRAS family proteins in Medicago truncatula.Acta Physiologiae Plantarum,39(4):93.
Su Li-yuan,Li Xiao-zhe,Chen Shu-wen,Zhang Fang-fang,Zhao Ting-ting,Yang Gui-yan.2018.Expression and function analysis of walnut JrGRAS2 gene under heat stress.Botanical Research,38(1):125-131.(in Chinese)粟莉圆,李孝哲,陈淑雯,张芳芳,赵婷婷,杨桂燕.2018.核桃JrGRAS2基因响应热胁迫的表达及功能分析.植物研究,38(1):125-131.
Sun T.2010.Gibberellin-GID1-DELLA:a pivotal regulatory module for plant growth and development.Plant Physiology,154(2):567-570.
Torres-Galea P,Hirtreiter B,Bolle C.2013.Two GRAS proteins,SCARECROW-LIKE21 and PHYTOCHROME a SIGNAL TRANSDUCTION1,function cooperatively in phytochrome a signal transduction.Plant Physiology,161(1):291-304.
Tyler L,Thomas S G,Hu J H,Dill A,Alonso J M,Ecker J R,Sun T P.2004.DELLA proteins and gibberellin-regulated seed germination and floral development in Arabidopsis.Plant Physiology,135(2):1008-1019.
Zhang Huan-xin,Dong Chun-juan,Shang Qing-mao.2017.Genome-wide identification and expression analysis of GRAS gene family in pepper.Acta Horticulturae Sinica,44(12):2305-2317.(in Chinese)张焕欣,董春娟,尚庆茂.2017.辣椒GRAS家族全基因组鉴定与表达分析.园艺学报,44(12):2305-2317.
Zhang Yu-xi,Zhang Wen-chao,Li Yu-e,Liu Chun-ying,Zheng Guo-sheng,Gai Shu-peng.2014.The study of PsGA20ox geneparticipating in endo-dormancy release of flower buds by chiling treatment in tree peony.Acta Agriculturae Boreali-Sinica,29(3):22-26.(in Chinese)张玉喜,张文超,李玉娥,刘春英,郑国生,盖树鹏.2014.赤霉素氧化酶PsGA20ox基因参与低温诱导的牡丹内休眠解除.华北农学报,29(3):22-26.
Zheng Guo-sheng,Gai Shu-peng,Gai Wei-ling.2009.Changes of endogenous hormones during dormancy release by chilling in tree peony.Scientia Silvae Sinicae,45(2):48-52.(in Chinese)郑国生,盖树鹏,盖伟玲.2009.低温解除牡丹芽休眠进程中内源激素的变化.林业科学,45(2):48-52.
Duan Chengguo,Li Xianli,Gao Dongsheng,Liu Huanfang,Li Meng.2004.Studies on regulations of endogenous ABA and GA3 in sweet cherry flower buds on dormancy.Acta Horticulturae Sinica,31(2):149-154.(in Chinese)段成国,李宪利,高东升,刘焕芳,李萌.2004.内源ABA和GA3对欧洲甜樱桃花芽自然休眠的调控.园艺学报,31(2):149-154.
Finn R D,Bateman A,Clements J,Coggill P,Eberhardt R Y,Eddy S R,Heger A,Hetherington K,Holm L,Mistry J,Sonnhammer E L L,Tate J,Punta M.2014.Pfam:the protein families database.Nucleic Acids Research,42:222-230.
Gai Shupeng,Zhang Feng,Zhang Yuxi,Zheng Guosheng.2012.Analysis of genomic DNA methylation during chilling induced endo-dormancy release by methylation sensitive amplified polymorphism(MSAP)technology in tree peony(Paeonia suffruticosa).Journal of Agricultural Biotechnology,20(3):261-267.(in Chinese)盖树鹏,张风,张玉喜,郑国生.2012.低温解除牡丹休眠进程中基因组DNA甲基化敏感扩增多态性(MSAP)分析.农业生物技术学报,20(3):261-267.
Gai S P,Zhang Y X,Liu C Y,Zhang Y,Zheng G S.2013.Transcript profiling of Paoenia ostii during artificial chilling induced dormancy release identifies activation of GA pathway and carbohydrate metabolism.PLoS ONE,8(2):e55297.
Gai S P,Zhang Y X,Mu P,Liu C Y,Liu S,Dong L,Zheng G S.2012.Transcriptome analysis of tree peony during chilling requirement fulfillment:assembling,annotation and markers discovering.Gene,497(2):256-262.
Huang X,Zhu W,Dai S L,Gai S P,Zheng G S,Zheng C C.2008.The involvement of mitochondrial phosphate transporter in accelerating bud dormancy release during chilling treatment of tree peony(Paeonia suffruticosa).Planta,228(4):545-552.
Koizumi K,Gallagher K L.2013.Identification of SHRUBBY,a SHORT-ROOT and SCARECROW interacting protein that controls root growth and radial patterning.Development,140(6):1292-1300.
Li W Q,Wu J G,Weng S L,Zhang Y J,Zhang D P,Shi C H.2010.Identification and characterization of dwarf 62,a loss-of-function mutation in DLT/OsGRAS-32 affecting gibberellin metabolism in rice.Planta,232(6):1383-1396.
Livak J,Schmittgen D.2001.Analysis of relative gene expression data using real time quantitative PCR and the 2-??Ct method.Methods,25(4):402-408.
Locascio A,Blázquez M A,AlabadíD.2013.Genomic analysis of DELLA protein activity.Plant&Cell Physiology,54(8):1229-1237.
Lu Jiu-xing.2015.Functional analysis of DELLA genes related to dormancy in Prunus mume[Ph.D.Dissertation].Beijing:Beijing Forestry University.(in Chinese)逯久幸.2015.梅花休眠相关DELLA基因的克隆与功能分析[博士论文].北京:北京林业大学.
Mornya P M P,Cheng F Y.2018.Effect of combined chilling and GA3 treatment on bud abortion in forced‘Luoyanghong’tree peony(Paeonia suffruticosa Andr.).Horticultural Plant Journal,4(6):250-256.
Morohashi K,Minami M,Takase H,Hotta Y,Hiratsuka K.2003.Isolation and characterization of a novel GRAS gene that regulates meiosis-associated gene expression.Journal of Biological Chemistry,278(23):20865-20873.
Pysh L D,Wysocka-Diller J W,Camilleri C,Benfey P N.1999.The GRAS gene family in Arabidopsis:sequence characterization and basic expression analysis of the SCARECROW-LIKE genes.Plant Journal for Cell&Molecular Biology,18(1):111-119.
Song Li-li,Tao Lei,Cui Hui-ping,Ling Lei,Guo Chang-hong.2017.Genome-wide identification and expression analasis of th-e GRAS family proteins in Medicago truncatula.Acta Physiologiae Plantarum,39(4):93.
Su Li-yuan,Li Xiao-zhe,Chen Shu-wen,Zhang Fang-fang,Zhao Ting-ting,Yang Gui-yan.2018.Expression and function analysis of walnut JrGRAS2 gene under heat stress.Botanical Research,38(1):125-131.(in Chinese)粟莉圆,李孝哲,陈淑雯,张芳芳,赵婷婷,杨桂燕.2018.核桃JrGRAS2基因响应热胁迫的表达及功能分析.植物研究,38(1):125-131.
Sun T.2010.Gibberellin-GID1-DELLA:a pivotal regulatory module for plant growth and development.Plant Physiology,154(2):567-570.
Torres-Galea P,Hirtreiter B,Bolle C.2013.Two GRAS proteins,SCARECROW-LIKE21 and PHYTOCHROME a SIGNAL TRANSDUCTION1,function cooperatively in phytochrome a signal transduction.Plant Physiology,161(1):291-304.
Tyler L,Thomas S G,Hu J H,Dill A,Alonso J M,Ecker J R,Sun T P.2004.DELLA proteins and gibberellin-regulated seed germination and floral development in Arabidopsis.Plant Physiology,135(2):1008-1019.
Zhang Huan-xin,Dong Chun-juan,Shang Qing-mao.2017.Genome-wide identification and expression analysis of GRAS gene family in pepper.Acta Horticulturae Sinica,44(12):2305-2317.(in Chinese)张焕欣,董春娟,尚庆茂.2017.辣椒GRAS家族全基因组鉴定与表达分析.园艺学报,44(12):2305-2317.
Zhang Yu-xi,Zhang Wen-chao,Li Yu-e,Liu Chun-ying,Zheng Guo-sheng,Gai Shu-peng.2014.The study of PsGA20ox geneparticipating in endo-dormancy release of flower buds by chiling treatment in tree peony.Acta Agriculturae Boreali-Sinica,29(3):22-26.(in Chinese)张玉喜,张文超,李玉娥,刘春英,郑国生,盖树鹏.2014.赤霉素氧化酶PsGA20ox基因参与低温诱导的牡丹内休眠解除.华北农学报,29(3):22-26.
Zheng Guo-sheng,Gai Shu-peng,Gai Wei-ling.2009.Changes of endogenous hormones during dormancy release by chilling in tree peony.Scientia Silvae Sinicae,45(2):48-52.(in Chinese)郑国生,盖树鹏,盖伟玲.2009.低温解除牡丹芽休眠进程中内源激素的变化.林业科学,45(2):48-52.