菊花AINTEGUMENTA克隆与功能分析  

Cloning and Functional Verification of AINTEGUMENTA Gene in Chrysanthemum

作　　者：温立柱[1] 孙霞[1] 樊红梅 郭芸珲 于媛媛[1] 任红 王文莉[1] 郑成淑[1] WEN LiZhu;SUN Xia;FAN HongMei;GUO YunHui;YU YuanYuan;REN Hong;WANG WenLi;ZHENG ChengShu(College of Horticulture Science and Engineering, Shandong Agricultural University~Chrysanthemum Research Center of China, Japan and Korea in Shandong Province, Tai ＇an 2 71018, Shandong)

机构地区：[1]山东农业大学园艺科学与工程学院/山东省中日韩菊花国际合作研究中心,山东泰安271018

出　　处：《中国农业科学》2018年第9期1771-1782,共12页Scientia Agricultura Sinica

基　　金：国家科技支撑计划(2011BAD10B07);国家自然科学基金面上基金(31670663)

摘　　要：【目的】从菊花中分离克隆AINTEGUMENTA,分析其序列特征和在菊花中的时空表达特性,通过基因沉默研究其对菊花花序发育的影响,分析可能的调控方式和潜在机理,为菊花花序大小的调控奠定理论基础。【方法】使用RACE方法从菊花中克隆AINTEGUMENTA全长,通过DNAMAN等软件对其序列进行分析。采用荧光定量的方式检测其在菊花不同器官和发育时期的表达。构建35S::CmANT-GFP融合蛋白载体进行亚细胞定位,构建TRV2-CmANT沉默表达载体侵染菊花。使用SPSS软件统计分析CmANT沉默系菊花表型变化,通过光学显微镜观察舌状花花瓣表皮细胞变化,使用荧光定量方式检测CmANT相关基因在沉默系中的表达。【结果】从菊花中克隆了CmANT全长,其编码的氨基酸序列长度为540,理论等电点为7.39,蛋白质的理论分子量为60.4 k D,含有两个AP2保守功能区域和VYL修饰位点。在与其他物种的ANT蛋白构建的系统进化树中,CmANT与At ANT聚在一起。荧光定量结果表明:(1)CmANT在花蕾中的表达量最高,其次是根>茎>叶。(2)在花序不同部位的比较中,舌状花中的表达量最高,其次是筒状花,在花萼中的表达量最低。(3)CmANT在舌状花中的表达随着发育时期的延续而降低。(4)CmANT在2,4-D诱导下的3—6 h内表达量持续上升。Wo LF PSORT软件预测和35S::CmANT-GFP融合蛋白在洋葱表皮细胞中的定位结果显示,CmANT蛋白定位在植物细胞核中。TRV-CmANT-1和TRV-CmANT-2沉默系的平均花径相比对照分别减小18.93%和27.47%,舌状花的数目分别减少11.39%和14.66%,其中TRV-CmANT-2与对照差异显著(P<0.05),筒状花数目分别减少14.55%和36.56%。顶端花序的舌状花平均长度分别减少34.17%和54.68%,舌状花的宽度分别比对照减小24.05%和10.13%。叶片平均鲜重分别比对照组减小13.19%和21.98%,叶片鲜重与筒状花数目显著相关(P<0.05)。舌状花花瓣表皮细胞显微观察发现,沉默系舌状【Objective】 To understand the role of AINTEGUMENTA gene in chrysanthemum inflorescence development, we cloned AINTEGUMENTA gene from chrysanthemum, analyzed its sequence, and characterized its temporal and spatial expression pattern. We further analyzed the impact of AINTEGUMENTA silence on inflorescence development and its possible regulation mode. This study was carried to reveal the potential mechanism of AINTEGUMENTA in governing inflorescence development, which in turn can provide a theoretical basis for chrysanthemum inflorescence diameter adjustment. 【Method】 The chrysanthemum AINTEGUMENTA gene was cloned by RACE method and its sequence was analyzed with DANMAN software. Its expression in different developmental stages and organs of chrysanthemum was detected by real-time fluorescent quantitation PCR. The plasmid of 35 S：：CmANT-GFP was constructed for the subcellular localization. The silence vector of TRV2-CmANT was constructed to infect chrysanthemums. The statistics of phenotypic changes in CmANT silenced chrysanthemums were analyzed via SPSS software. The ray florets petal epithelial cells were observed with optical microscope. The expression of CmANT and related genes were detected by real-time fluorescent quantitation PCR. 【Result】 The full-length of CmANT was cloned from chrysanthemum. It encodes 540 amino acids and contains two AP2 conserved function domains and VYL modification sites. The theoretical isoelectric point of CmANT is 7.39 and its molecular weight is 60.4 k D. The phylogenetic tree composed of ANT proteins from various plants species showed that CmANT and Aa ANT had been grouped together. The real-time fluorescent quantitation results showed that：（1） CmANT was expressed most in floral buds, followed by roots, stems and leaves.（2） Gene expression in different parts of inflorescence indicated that CmANT was expressed the most in ray florets followed tubular florets and the lowest in the sepal.（3） The expression of CmANT declined during the development of ray

关 键 词：菊花 AINTEGUMENTA 花径 基因表达 基因沉默 

分 类 号：S682.11[农业科学—观赏园艺]