HE Jingzhou,FAN Jizheng,ZENG Yanhua,et al.Molecular Cloning and Expression of APETALA1 Gene From Phalaenopsis ‘Big Chili’[J].Northern Horticulture,2021,(03):83-90.[doi:10.11937/bfyy.20200592]
蝴蝶兰“大辣椒”APETALA1基因的克隆及表达
- Title:
- Molecular Cloning and Expression of APETALA1 Gene From Phalaenopsis ‘Big Chili’
- Keywords:
- Phalaenopsis; AP1 gene; gene expression
- 文献标志码:
- A
- 摘要:
- 以蝴蝶兰“大辣椒”为试材，采用RACE技术克隆蝴蝶兰APETALA1（AP1）基因的cDNA全长,分析基本生物学信息，并初步探索其在花芽分化过程中的作用。结果表明：AP1基因的cDNA全长为1 155 bp，开放阅读框(open reading frame)752 bp，编码250个氨基酸，含有MADS盒与K盒等保守功能结构域。同源性比对结果显示，蝴蝶兰“大辣椒”与朵丽蝶兰、金蝶兰、文心兰、石斛兰和马兜铃等植物的MADS蛋白有较高的相似性，同源性均在80%以上；系统进化树分析显示，蝴蝶兰“大辣椒”AP1基因与朵丽蝶兰聚类关系最近；实时荧光定量PCR检测发现，AP1基因在叶片、根和花葶中均有表达，但表达量有差异。同一器官不同发育时期比较显示，叶片的AP1基因表达量没有明显的时空差异；根AP1基因的表达量随着花芽分化的进程呈递减趋势，其中始花期的表达量最低，盛花期最高；而花葶中AP1基因表达量随着花芽分化呈增加趋势，在盛花期表达量达到峰值。同一时期不同器官比较，花葶中AP1的表达量显著高于同一时期的叶片和根（P<0.05）。由此，推测AP1基因在调控蝴蝶兰花芽分化过程中发挥重要作用。
- Abstract:
- Taking Phalaenvpsis ‘Big Chili’ as experiment material,cloning full length cDNA of Phalaenopsis APETALA1 gene by RACE and analysis of basic biological infamati.The results showed full-length cDNA of AP1 gene was 1 155 bp with a 752 bp open reading frame encoding a putative protein of 250 amino acids.It contained conservative functional domains such as MADS-box and K-box.The results of homology comparison showed that the MADS proein of Phalaenopsis ‘Big Chili’ had high similarity with other plants,such as Dory butterfly,Cymbidium formosans,Oncidium,Dendrobium and Aristolochia,and the homology was more than 80%.Phylogenetic tree analysis showed that the AP1 gene of Phalaenopsis ‘Big Chili’ had the closest relationship with the clustering of Dory butterfly.qRT-PCR showed that the mRNA of AP1 was widely expressed in all tissues tested (leaves,roots and pedicels),but the expression level was different.There was no signficant difference of AP1 gene expression in leaves of different developmental stages of the same organ.The expression level of AP1 gene in roots decreased with the process of flower bud differentiation,the lowest level at the beginning flowering stage,and the highest at the full flowering stage;while the expression of AP1 gene in the scape increased with the flower bud differentiation,and the highest at the full flowering stage.Comparison of different organs in the same period,the expression of AP1 in pedicel was significantly higher than that in leaves and roots (P<0.05).Therefore,we speculate that AP1 gene plays an important role in the regulation of flower bud differentiation in Phalaenopsis.
参考文献/References:
[1]俞继英,张阳,郑锦凯,等.蝴蝶兰黄花系品种Phalaenopsis ‘Tai Gold’和P.‘Sara Gold’杂交及其后代的遗传表现[J].浙江林学院学报,2010,27(4):550-553.[2]TEIXEIRA D A,SILVA J A,CHIN D P,et al.Transgenic orchids[J].Scientia Horticulturae,2011,130(4):673-680.[3]KAUFMANN K,WELLMER F,MUIO J M,et al.Orchestration of floral initiation by APETALA1[J].Science,2010,328:85-89．[4]WELLMER F,ALVES-FERREIRA M,DUBOIS A,et al.Genomewide analysis of gene expression during early Arabidopsis flower development[J].PLoS Genetics,2006,2(7):e117.[5]JAYA E S,CLEMENS J,SONG J，et al.Quantitative expression analysis of meristem identity genes in Eucalyptus occidentalis:AP1 is an expression marker for flowering[J].Tree Physiology,2010,30(2):304-312．[6]PABN-MORA N,AMBROSE B A,LITT A.Poppy APETALA1/FRUITFULL orthologs control flowering time,branching,perianth identity,and fruit development[J].Plant Physiology,2012,158(4):1685-1704．[7]FERNANDO D D,ZHANG S L.Constitutive expression of the SAP1 gene from willow (Salix discolor) causes early flowering in Arabidopsis thaliana[J].Development Genes and Evolution,2006,216(1):19-28.[8]PENA L,TRILLO M M,JUAREZ J,et al.Constitutive expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time[J].Nature Biotechnology,2001,19:263-267.[9]GE J,ZHAO D,HAN C,WANG J,et al.Cloning and expression of floral organ development-related genes in herbaceous peony (Paeonia lactiflora Pall.)[J].Mol Biol Rep,2014,41(10):6493-6503.[10]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408.[11]VANDENBUSSCHE M,THEIEN G,van DE PEER Y,et al.Structural diversification and neo-function alization during floral MADS-box gene evolution by C-terminal frame shift mutations[J].Nucleic Acids Res,2003,31:4401-4409.[12]高永霞.蝴蝶兰花期调控基因的克隆及功能研究[D].南京:南京农业大学,2017.[13]THOMAS J.Molecular and genetie mechanisms of floral control[J].Plnat Cell,2004,16(S):1-17.[14]刘艺平,董姬秀,张曼等.荷花LEAFY基因的克隆及表达分析[J].植物生理学报,2014,50(2):203-208.[15]COUPLAND G.LEAFY blooms in aspen[J].Nature,1995,377:482-483.[16]WICKLAND D P,HANZAWA Y.The Flowering Locus T/Terminal Flower 1 gene family:Functional evolution and molecular mechanisms[J].Mol Plant,2015,8(7):983-997.[17]罗火林,张亚楠,罗丽萍,等.兰科植物FT基因的密码子偏性分析[J].分子植物育种,2016,14(1):51-58.[18]KAUFMANN K,NAGASAKI M,JAUREGUI R.Modelling the molecular interactions in the flower development network of Arabidopsis thaliana[J].Studies in health technology and informatics,2011,162:279-297.[19]GUTTERSON N,REUBER T L.Regulation of disease resistance pathways by AP2/ERF transcription factors[J].Current Opinion in Plant Biology,2004,7(4):465-471.[20]崔波,郝平安,梁芳,等.蝴蝶兰AP2/ERF家族基因的克隆及在低温下表达特性分析[J].园艺学报,2019(46):1-13.[21]董姬秀.荷花APETALA3、LEAFY基因的克隆与表达分析[D].郑州:河南农业大学,2010.[22]蒋素华,黄萍,王默霏,等.萼脊兰MADS-box基因的克隆及表达载体构建[J].华北农学报,2017,32(3):65-69.[23]葛春艳,李雪彤,刘福鹏,等．红景天AG基因(RrAG)的电子克隆及生物信息学分析[J]．江苏农业科学,2018,46(21):52-57.[24]THOMAS J.Molecular and genetic mechanisms of floral control[J].Plant Cell,2004,16:1-17.[25]MANDEL M A,GUSTAFSON-BROWN C,SAVIDGE B,et al.Molecular characterization of the Arabidopsis floral homeotic gene APETALA1[J].Nature,1992,360:273-277.[26]GE J,ZHAO D,HAN C,et al.Cloning and expression of floural organ development-related genes in herbaceous peony (Paeonia lactiflora Pall.)[J].Molecular Biology Reports,2014,41(10):6493-6503[27]WEIGEL D,NILSSON O.A developmental switch sufficient for flower initiation in diverse plants[J].Nature,1995,377(6549):495-500.[28]ELLUL P,ANGOSTO T,GARCIA-SOGO B,et a1.Expression of Arabidopsis APETALA1 in tomato reduces its vegetative cycle without affecting plant production[J].Molecular Breeding,2004,13(2):155-163.[29]YANG Y.Defining subdomains of the K domain important for protein-protein interactions of plant MADS proteins[J].Plant Molecular Biology,2004,55:45-59.[30]TSAI C C,CHIANG Y C,HUANG S C,et al.Molecular phylogeny of Phalaenopsis blume (Orchidaceae) on the basis of plastid and nuclear DNA[J].Plant Syst Evol,2010,288(1):77-98．[31]GRAMZOW L,THEISSEN G.A hitchhiker′s guide to the MADS world of plants[J].Genome Biol,2010,11(6):214．[32]CHANG Y Y,CHIU Y F,WU J W,et al.Four orchid（Oncidium Gower Ramsey）AP1/AGL9-like MADS box genes show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis thaliana[J].Plant Cell Physiol,2009,50:1425-1438.[33]张国付.萼脊兰AP1(APETALA1)基因的序列分析及表达特性研究[D].郑州:河南农业大学,2013[34]WU J H,WU S H,YANG C,et al.Cloning and expression analysis on AP1 homologous gene from Cymbidium ensifolium and interaction analysis between AP1 and MADS-box transcription factors[J].Acta Horticulturae Sinica,2013,40(10):1935-1942.[35]CUI B,WU Z J,LIU J,et al.Clonging and expression analysis of on AP1-like gene from oncidium ‘gower ramsey’[J].Acta Horticulturae Sinica,2014,41(2):357-364.[36]YUAN X Y,JIANG S H,TIAN Y F,et al.Cloning and expression analysis of a MADS-box gene from orichid[J].Journal of Henan Agricultural University,2013,47(6):683-690.[37]KIM S,KOH J,MA H,et al.Sequence and expression studies of A-,B-,and E-class MADS-box homologues in Eupomatia (Eupomatiaceae):Support for the bracteate origin of the calyptra[J].International Journal of Plant Sciences,2005,66:185-198.[38]BURKO Y,SHLEIZER-BURKO S,YANAI O,et al.A role for APETALA1/FRUITFULL transcription factors in tomato leaf development[J].Plant Cell,2013,25(6):2070-2083.[39]FORNARA F,PARENICOVA L,FALASCA G,et al.Functional characterization of Os MADS18,a member of the AP1/SQUA subfamily of MADS box genes[J].Plant Physiology,2004,135:2207-2219.
相似文献/References:
[1]孙汝斌,余 纽,孙佩光.蝴蝶兰基因工程研究进展[J].北方园艺,2014,38(12):177.
SUN Ru-bin,YU Niu,SUN Pei-guang.Research Progress on Gene Engineering of Phalaenopsis Plants[J].Northern Horticulture,2014,38(03):177.
[2]董平,赵培宝,任爱芝.蝴蝶兰灰霉病菌的分离鉴定[J].北方园艺,2014,38(04):109.
DONG Ping,ZHAO Pei-bao,REN Ai-zhi.Isolation and Identification of Botrytis cinerea?in Phalaenopsis[J].Northern Horticulture,2014,38(03):109.
[3]王常芸,李晓亮,张京伟,等.蝴蝶兰组培快繁技术体系研究[J].北方园艺,2013,37(15):122.
WANG Chang-yun,LI Xiao-liang,ZHANG Jing-wei,et al.Study on Tissue Culture and Rapid Propagation System of Phalaenopsis[J].Northern Horticulture,2013,37(03):122.
[4]丁兰,郭艳,董刚,等.国产蝴蝶兰种苗携带建兰花叶病毒(CymMV)和齿兰环斑病毒 (ORSV)的调查及脱毒的初步研究[J].北方园艺,2012,36(02):137.
DING Lan,GUO Yan,DONG Gang,et al.Investigation and Preliminary Study of Elimination of CymMV and ORSV from Phalaenopsis in China[J].Northern Horticulture,2012,36(03):137.
[5]孔芳,凌宏龙,薛正莲,等.保鲜液对蝴蝶兰切花保鲜生理效应的研究[J].北方园艺,2013,37(02):130.
KONG Fang,LING Hong-long,XUE Zheng-lian,et al.Research on Preservatives of Cut Phalaenopsis Flower and Related Physiological Influence[J].Northern Horticulture,2013,37(03):130.
[6]景维杰,杨露露,蒋明殿.不同浓度氮、磷、钾对蝴蝶兰幼苗生长的影响[J].北方园艺,2012,36(23):62.
JING Wei-jie,YANG Lu-lu,JIANG Ming-dian.Effects of Different Concentrations of Nitrogen，Phosphorus and Potassium on the Growth of Phalaenopsis Seedlings[J].Northern Horticulture,2012,36(03):62.
[7]何荆洲,卜朝阳,黄昌艳,等.蝴蝶兰红花系品种“火凤凰”的杂交结实性研究[J].北方园艺,2015,39(02):61.[doi:10.11937/bfyy.201502017]
HE Jing-zhou,BU Zhao-yang,HUANG Chang-yan,et al.Study on Fecundity of Cross-pollination in Phalaenopsis ‘Fire Phoenix’[J].Northern Horticulture,2015,39(03):61.[doi:10.11937/bfyy.201502017]
[8]杜君,王利民,孟月娥,等.我国蝴蝶兰产业发展现状及对策研究[J].北方园艺,2015,39(09):172.[doi:10.11937/bfyy.201509047]
DU Jun,WANG Li-min,MENG Yue-e,et al.Study on the Development Status and Countermeasures of Phalaenopsis amabilis Industry in China[J].Northern Horticulture,2015,39(03):172.[doi:10.11937/bfyy.201509047]
[9]张 颖,郭 晓 东,王芳,等.蝴蝶兰组织培养快繁技术研究[J].北方园艺,2010,34(08):0.[doi:10.11937/bfyy.201008046]
ZHANG Ying,GUO Xiao -dong,W ANG Fang,et al.The Research on Tissue Culture and Rapid Propagation of Phalaenopsis Hybrid[J].Northern Horticulture,2010,34(03):0.[doi:10.11937/bfyy.201008046]
[10]王 桂 兰,陈 超,李 艳 梅,等.蝴蝶兰催花及开花过程中可溶性糖含量变化的研究[J].北方园艺,2010,34(10):0.[doi:10.11937/bfyy.2010010055]
W ANG Gui -lan,CHEN Chao,LI Yan -mei,et al.Study on Change of Soluble Sugar Content in Plant of Phalaenopsis During Flower Induction and Blooming Period[J].Northern Horticulture,2010,34(03):0.[doi:10.11937/bfyy.2010010055]
备注/Memo
第一作者简介：何荆洲（1981-），女，硕士，副研究员，现主要从事生物技术与植物育种等研究工作。E-mail:he5141@sina.com.责任作者：卜朝阳（1966-），女，硕士，研究员，现主要从事花卉育种等研究工作。E-mail:yangnv@126.com.基金项目：广西科技重大专项资助项目（桂科AA17204046-1）；广西科技基地和人才专项资助项目（桂科AD17129023）；广西自然基金资助项目（2020GXNSFAA159044）;广西农业科学院团队资助项目（2015YT90）。收稿日期：2020-02-20