甘菊ClCOL16基因的克隆与功能初步研究  

Cloning and Functional Analysis of ClCOL16 from Chrysanthemum lavandulifolium

作　　者：陈新娜 张利英 喻锌 陈东亮[2,3] 黄丛林 李彦慧[1] CHEN Xinna;ZHANG Liying;YU Xin;CHEN Dongliang;HUANG Conglin;LI Yanhui(College of Landscape and Tourism,Hebei Agricultural University,Baoding,Hebei 071000,China;Institute of Grassland,Flowers and Ecology,Beijing Academy of Agriculture and Forestry Sciences,Beijing 100097,China;Beijing Functional Flower Engineering Technology Research Center,Beijing 100097,China)

机构地区：[1]河北农业大学园林与旅游学院,河北保定071000 [2]北京市农林科学院草业花卉与景观生态研究所,北京100097 [3]北京市功能花卉工程技术研究中心,北京100097

出　　处：《西北植物学报》2023年第3期366-373,共8页Acta Botanica Boreali-Occidentalia Sinica

基　　金：北京市农林科学院青年基金(QNJJ202010);北京市农林科学院创新能力建设专项(KJCX20200106)。

摘　　要：CO基因通过光周期途径整合昼夜规律、光信号以及分生组织相关基因来调节开花时间,在植物成花过程中起着至关重要的作用。该研究利用RT-PCR法从甘菊(Chrysanthemum lavandulifolium)中克隆得到一个CO家族基因,命名为ClCOL16(GenBank登录号:AOA52174);采用农杆菌介导的花序浸染法转化拟南芥,抗性筛选并经过PCR鉴定得到T3代转基因植株;随机抽取6个转基因拟南芥株系与野生型(WT)共同于长日照环境(16 h/8 h光暗交替)培养,观察其表型,并于定植后37 d检测WT和转基因拟南芥植株叶片的ClCOL16表达,以明确甘菊ClCOL16基因在植物光周期调控开花中的作用,为进一步解析菊花花期调控机理奠定基础。结果显示:(1)甘菊ClCOL16基因含有一个1 278 bp的编码框,编码425个氨基酸;ClCOL16编码蛋白含有一个B-box和一个CCT保守结构域,为典型的CO家族第Ⅲ组成员。(2)NCBI同源比对分析显示,ClCOL16蛋白与除虫菊COL16蛋白(GenBank登录号:GEZ38716.1)的一致性最高,达到94.38%。(3)荧光定量分析显示,ClCOL16基因为组成型表达,在叶片中的表达量最高,其表达受生物钟调节,在短日照调控下表现出明显的昼夜节律。(4)异位表达结果显示,与野生型拟南芥相比,转ClCOL16基因拟南芥株系的花期提前了7 d,莲座叶减少了3~4片,叶片中ClCOL16基因的表达量均显著提高,表明ClCOL16基因能够促进转基因拟南芥开花。研究推测,甘菊ClCOL16基因可能是甘菊响应光信号、参与菊花光周期开花途径调控的重要基因。The CO family genes regulate flowering time by integrating circadian rhythms,light signals,and meristem-related genes through the photoperiod pathway,and play a crucial role in the process of plant flower formation.In this study,a CO family gene cloned from Chrysanthemum lavandulifolium by RT-PCR method,was named ClCOL16(GenBank accession number:AOA52174.1).Arabidopsis thaliana was transformed by inflorescence immersion mediated by Agrobacterium tumefaciens,and T3 generation transgenic plants were selected by resistant screening and PCR detection of the heterologous COL gene.To analysis the function of ClCOL16 in plant photoperiod regulation of flowering and lay the foundation for the further study for flowering regulation mechanism in Chrysanthemum morifolium,we cultured six transgenic lines randomly selected and the wild type(WT)of A.thaliana in a long day environment(alternating light and dark at 16 h/8 h)to observe their phenotypes,and detected the expression of ClCOL16 in WT and transgenic A.thaliana leaves after the 37 days of planting.The results showed:(1)ClCOL16 gene of C.lavandulifolium ORF was 1278 bp and encoding a protein of 425 amino acids;Structural analysis showed ClCOL16 protein contains a B-box and a CCT conserved domain,which is a typical characteristic of CO family GroupⅢ.(2)NCBI blast analysis showed that ClCOL16 protein had the highest identity with COL16(GenBank accession number:GEZ38716.1)from Tanacetum cinerariifolium,up to 94.38%,which further proved that it belongs to the CO family GroupⅢ.(3)Expression analysis showed that ClCOL16 gene was constitutive expression in C.lavandulifolium,and the highest expression was in leaves;ClCOL16 gene was regulated by circadian clock,under short-day condition its expression showing obvious circadian rhythm.(4)Ectopic expression showed that compared to wild type A.thaliana,the flowering time of ClCOL16 transgenic lines was advanced by 7 days,the number of rosette leaves was reduced by 3 to 4,and the expression of ClCOL16 gene in leaves was signific

关 键 词：甘菊 COL16 花期调控 异位表达 

分 类 号：Q785[生物学—分子生物学] Q786Q789