菊花AINTEGUMENTA克隆与功能分析

doi:10.3864/j.issn.0578-1752.2018.09.014

摘要/Abstract

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 kD，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AtANT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。WoLF 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，沉默系舌状花花瓣表皮细胞长度和宽度与对照差异不明显。CmLAX3在两沉默系中的表达明显上升，在小花原基分化期时分别是对照中的1.8和1.78倍，同期CmCYCD3的表达分别下降了32.28%和38.19%，CmXTH4和CmEXPA1的表达量在多个时期也明显降低。【结论】根据沉默系表型与相关基因的表达，推测CmANT的沉默可能解除了对CmLAX3抑制，促进了生长素的转运和过量积累，间接抑制了CmCYCD3的活性，限制了细胞的分裂增殖，引发细胞数目变少，导致沉默系器官变小。

关键词: 菊花, AINTEGUMENTA, 花径, 基因表达, 基因沉默

Abstract: 【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 35S::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 kD. The phylogenetic tree composed of ANT proteins from various plants species showed that CmANT and AaANT 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 florets. (4) The expression of CmANT under 2,4-D treatment increased between 3 h to 6 h. WoLF PSORT prediction and 35S::CmANT-GFP fusion protein localization in onion epithelial cells indicated that CmANT protein was located in cell nucleus. Compared with the control, the mean inflorescence diameters of chrysanthemums in TRV-CmANT-1 and TRV-CmANT-2 lines were decreased by 18.93% and 27.47% respectively and the numbers of ray florets were decreased by 11.39% and 14.66% respectively, among which the difference between TRV-CmANT-2 lines and the control was statistically significant (P＜0.05 ). The numbers of tubular florets were decreased by 14.55% and 36.56%, the mean length of ray florets in top inflorescences was decreased by 34.17% and 54.68%, and the width was decreased by 24.05% and 10.13% respectively in TRV-CmANT-1 and TRV-CmANT-2 lines compared with the control, the mean fresh weight of leaves was decreased by 13.19% and 21.98% respectively in TRV-CmANT-1 and TRV-CmANT-2 lines and the correlation between leaf fresh weight and tubular floret number was significant (P＜0.05 ). The microscopic observation of epithelial cells in ray floret petals indicated that the cell length and width of petals in the silenced lines and the control had no visible differences. The expression of CmLAX3 in two silenced chrysanthemums lines were 1.8 and 1.78 times higher than the control, respectively. Meanwhile the expression of CmCYCD3 was decreased by 32.28% and 38.19% in two silenced lines and the expression of CmXTH4 and CmEXPA1 also decreased at most developmental stages. 【Conclusion】 Due to the phenotypic changes and CmANT expression pattern in silenced chrysanthemums lines, we speculated that the silence of CmANT might relieve the repression on CmLAX3, facilitate the transport and accumulation of auxins, indirectly repress the activation of CmCYCD3, limit the cell division, and cause the decrease of cell number, combined of which would eventually lead to the smaller organ sizes in silenced chrysanthemums lines.

Key words: Chrysanthemum morifolium, AINTEGUMENTA, inflorescence diameter, gene expression, gene silence

温立柱，孙霞，樊红梅，郭芸珲，于媛媛，任红，王文莉，郑成淑. 菊花AINTEGUMENTA克隆与功能分析[J]. 中国农业科学, 2018, 51(9): 1771-1782.

WEN LiZhu, SUN Xia, FAN HongMei, GUO YunHui, YU YuanYuan, REN Hong, WANG WenLi, ZHENG ChengShu. Cloning and Functional Verification of AINTEGUMENTA Gene in Chrysanthemum[J]. Scientia Agricultura Sinica, 2018, 51(9): 1771-1782.

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