菊花CmETR2基因的克隆及功能分析  

Cloning and functional analysis of CmETR2 gene in chrysanthemum

作　　者：周敏 程华 张子昕 邢晓娟 蒋甲福[1] 陈发棣[1] ZHOU Min;CHENG Hua;ZHANG Zixin;XING Xiaojuan;JIANG Jiafu;CHEN Fadi(College of Horticulture/Key Laboratory of Landscaping Agriculture,Ministry of Agriculture and Rural Affairs,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学园艺学院/农业农村部景观农业重点实验室,江苏南京210095

出　　处：《南京农业大学学报》2020年第3期431-437,共7页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31572159);江苏现代农业产业技术体系建设项目[JATS(2019)421]。

摘　　要：[目的]本文旨在通过克隆菊花中的乙烯受体基因(CmETR2),分析其表达特征并转入拟南芥中对其调控开花的功能进行研究。[方法]以菊花品种‘神马’为试验材料,克隆CmETR2全长序列,与其他物种进行同源性比对分析,利用荧光定量PCR检测该基因在不同组织中的相对表达量,以及其对外源乙烯处理后的响应变化。将CmETR2超表达转入拟南芥中,观察其对拟南芥开花时间和莲座叶片数的影响,分析转基因拟南芥中开花相关基因的表达量变化。[结果]克隆获得的CmETR2基因开放阅读框(ORF)全长为2292 bp,编码氨基酸763个。系统进化分析表明,该基因编码的蛋白与向日葵的ETR3(XP_022030036.1)和刺菜蓟的ETR2-like(XP_024986908.1)亲缘关系最近。组织特异性定量表达分析结果表明,CmETR2在菊花营养生长期间茎和叶中表达量最高,生殖生长期间管状花和根中的表达量较高。亚细胞定位结果表明,CmETR2定位在细胞膜上。外源乙烯处理后3 h CmETR2基因表达量最高。CmETR2超表达植株的开花时间比野生型拟南芥晚3~4 d,开花时莲座叶片数比野生型拟南芥多3片左右。CmETR2超表达植株对乙烯的敏感性增强并表现出更明显的三重反应。在CmETR2超表达植株中,促进开花的基因AtGI、AtSPL3和AtFD在CmETR2超表达植株中的表达量较野生型下降,抑制开花的基因AtFLC和AtTFL在CmETR2超表达植株的表达量上调。[结论]本研究克隆得到菊花CmETR2基因具有延迟拟南芥开花的功能。[Objectives]The purpose of this study was to clone the ethylene receptor gene of Chrysanthemum morifolium(CmETR2),analyze its expression characteristics and transfer it into Arabidopsis thaliana.A preliminary study was conducted on the function of regulating flowering.[Methods]The full-length sequence of CmETR2 was cloned from the chrysanthemum variety‘Jinba’and compared with other species’s CmETR2 sequences for homology alignment.Simultaneously,real-time quantitative PCR was used to analyze the relative expression level of the gene in different tissues.The samples were taken after exogenous ethylene treatment to observe its changes of response.CmETR2 gene was transferred into A.thaliana,and its effects on the flowering time and number of rosette leaves in A.thaliana were observed,and the expression level changes of related flowering genes in CmETR2 transgenic A.thaliana were analyzed.[Results]The full-length sequences of the open reading frame(ORF)of CmETR2 gene was 2292 bp,encoding 763 amino acids.The phylogenetic analysis indicated that the encoded protein was related to ETR3 in Helianthus annuus(XP_022030036.1)and ETR2-like in Cynara cardunculus(XP_024986908.1).Quantitative analysis of specific tissues showed that CmETR2 had the highest expression level in stems and leaves during vegetative growth,and the expression level in tubular flowers and roots was higher during reproductive growth.Subcellular localization result showed that CmETR2 was localized on the cell membrane.The expression level of CmETR2 was the highest at 3 h after exogenous ethylene treatment.The flowering time and number of rosette leaves of CmETR2 overexpression plants were observed and counted.The flowering time of CmETR2 overexpression plants was 3-4 days later than that of wild-type,and the number of rosette leaves was more than that of wild-type.The sensitivity of CmETR2 overexpression plants to ethylene was enhanced and more apparent in triple response.In CmETR2 overexpression plants,the expression levels of AtGI,AtSPL3 and AtFD

关 键 词：菊花 CmETR2基因 乙烯受体 开花 

分 类 号：S682.1[农业科学—观赏园艺]