菊花CmNFYC4促进开花的功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2026-0324

摘要/Abstract

摘要：

目的核因子Y（nuclear factor Y）是一种普遍存在于真核生物中的转录因子，其在植物胚胎发育、光合作用、开花调控、逆境胁迫等多方面有重要作用。克隆菊花（Chrysanthemum morifolium ‘Jinba’）CmNFYC4基因、分析其表达模式并进行功能验证，为菊花开花调控分子机制研究奠定基础。方法以菊花‘神马’为试验材料，克隆CmNFYC4的编码区序列。利用生物信息学技术对该蛋白的理化性质进行分析预测；同时采用荧光定量PCR技术检测该基因在菊花各组织中及在不同光照处理下基因表达量的变化。利用烟草瞬时表达系统研究该基因的亚细胞定位，并采用酵母双杂交技术对CmNFYC4的转录激活活性进行验证。构建融合抑制子沉默载体，通过农杆菌介导的叶盘侵染法，结合菊花转基因体系获得沉默株系；利用菊花瞬时转化体系创制CmNFYC4过表达株系，并进行开花表型观察。结果 CmNFYC4开放阅读框（ORF）全长726 bp，编码241个氨基酸，相对分子质量为26.48 kD，理论等电点（pI）为5.35。系统进化发育树分析表明，CmNFYC4蛋白与拟南芥和甘蓝型油菜的NFYC类基因亲缘关系较近，且具有较高的保守性。烟草亚细胞定位试验表明CmNFYC4定位于细胞核，酵母双杂交试验验证CmNFYC4具有转录激活活性。荧光定量PCR数据分析显示，CmNFYC4在菊花‘神马’各组织中均有表达，且在叶片中表达量最高。创制CmNFYC4融合抑制子沉默株系并进行表型观察，发现具有晚花表型，同时瞬转CmNFYC4过表达株系具有早花表型。结论 CmNFYC4在菊花叶片中表达量最高，其沉默株系具有晚花表型，过表达株系则表现为早花，表明CmNFYC4具有促进菊花开花的功能。

关键词: 菊花, CmNFYC4, 开花调控, 表达模式, 亚细胞定位, 转录激活, 转基因沉默, 瞬时过表达

Abstract:

Objective Nuclear factor Y (NFY) is a ubiquitous transcription factor in eukaryotes that plays crucial roles in multiple biological processes in plants, including embryo development, photosynthesis, flowering regulation, and stress response. This study aimed to clone the CmNFYC4 gene from Chrysanthemum morifolium ‘Jinba’ and systematically characterize its expression pattern and function, thereby providing a basis for investigating the molecular mechanisms underlying flowering regulation in C. morifolium. Methods Using C. morifolium ‘Jinba’ as the experimental material, the coding sequence (CDS) of CmNFYC4 was amplified. Bioinformatics tools were used to predict physicochemical properties. Quantitative real-time PCR (RT-qPCR) was performed to detect the expression patterns of CmNFYC4 in different tissues and its expression changes under various light treatments. Subcellular localization of CmNFYC4 was analyzed using a tobacco transient expression system. Transcriptional activation activity was verified using the yeast two-hybrid system. To investigate the biological function of CmNFYC4 in flowering regulation, we constructed a CmNFYC4-SRDX fusion repressor silencing vector and generated silencing lines of C. morifolium via Agrobacterium-mediated leaf disc transformation. Meanwhile, CmNFYC4 overexpression lines were created using a chrysanthemum transient transformation system, and the flowering phenotypes of transgenic plants were systematically observed and analyzed. Results Sequence analysis showed that the open reading frame (ORF) of CmNFYC4 was 726 bp, encoding 241 amino acids. The CmNFYC4 protein had a calculated molecular mass of 26.48 kD and a theoretical isoelectric point (pI) of 5.35. Amino acid sequence alignment and phylogenetic tree analysis revealed that CmNFYC4 was closely related to NFYC homologs from Arabidopsis thaliana and Brassica napus, indicating high conservation among NFYC family proteins. Subcellular localization assays demonstrated that CmNFYC4 was localized in the nucleus. A yeast two-hybrid experiment confirmed that CmNFYC4 possessed transcriptional activation activity. RT-qPCR results indicated that CmNFYC4 was ubiquitously expressed in all tested tissues, with the highest expression level in leaves. Phenotypic observation showed that, compared with wild-type plants, CmNFYC4-SRDX-silenced transgenic C. morifolium exhibited a delayed flowering phenotype, whereas CmNFYC4 overexpression lines displayed an early flowering phenotype. Conclusion CmNFYC4 is highly expressed in chrysanthemum leaves. Silencing lines exhibit a late-flowering phenotype, while overexpression lines show an early-flowering phenotype, indicating that CmNFYC4 functions as a promoter of flowering in chrysanthemum.

Key words: Chrysanthemum morifolium, CmNFYC4, flowering regulation, expression pattern, subcellular localization, transcriptional activation, transgenic silencing, transient overexpression

卢可新, 殷梦茹, 户倩, 蒋甲福. 菊花CmNFYC4促进开花的功能研究[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0324.

LU Ke-xin, YIN Meng-ru, HU Qian, JIANG Jia-fu. Functional Study of CmNFYC4 in Promoting Flowering of Chrysanthemum morifolium[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0324.

图/表 5

图1 CmNFYC4氨基酸序列系统发育树分析A：不同物种间NFYC蛋白的进化树分析；B：CmNFYC4蛋白与其他物种的氨基酸序列比对；AtNFYC1/AtNFYC4/AtNFYC10/AtNFYC11/AtNFYC13/AtNFYC12/AtNFYC6/AtNFYC7/AtNFYC8/AtNFYC5/AtNFYC2/AtNFYC9/AtNFYC3：拟南芥，AT3G48590/AT5G63470/AT1G07980/AT3G12480/AT5G43250/AT5G38140/AT5G50480/AT5G50470/AT5G27910/AT5G50490/AT1G56170/AT1G08970/AT1G54830；BnNFYC4：甘蓝型油菜，BnaA03g29270D；CmNFYC1：菊花，evm.TU.scaffold_223.199；CnNFYC1：菊花脑，Cn1014650；CsNFYC1：甘野菊，CsG_LG8.g62859；ZmNFYC4：玉米，Zm00001d020874；PhNFYC4：矮牵牛，Peaxi162Scf00102g01519；ASM3414082v1：粳稻，ASM3414082v1；HaNFYC4：向日葵，LOC110935072；CiNFYC4：野菊，CHR00079409-RA；StNFYC4.1/StNFYC4.2：马铃薯，XP_006351565/XP_006351565.1；DcNFYC1：铁皮石斛，DOG01G001230；OsHAP5C：水稻，Os03g0252000；GmNFYC4：大豆，06G169600

Fig. 1 Phylogenetic tree analysis of CmNFYC4 amino acid sequencesA: Phylogenetic tree analysis of NFYC proteins among different species. B: Amino acid sequence alignment of CmNFYC4 protein with other species. AtNFYC1/AtNFYC4/AtNFYC10/AtNFYC11/AtNFYC13/AtNFYC12/AtNFYC6/AtNFYC7/AtNFYC8/AtNFYC5/AtNFYC2/AtNFYC9/AtNFYC3: Arabidopsis thaliana, AT3G48590/AT5G63470/AT1G07980/AT3G12480/AT5G43250/AT5G38140/AT5G50480/AT5G50470/AT5G27910/AT5G50490/AT1G56170/AT1G08970/AT1G54830. BnNFYC4: Brassica napus L., BnaA03g29270D. CmNFYC1: C. morifolium, evm.TU.scaffold_223.199. CnNFYC1: Chrysanthemum nankingense, Cn1014650. CsNFYC1: C. seticuspe, CsG_LG8.g62859. ZmNFYC4: Zea mays ssp., Zm00001d020874. PhNFYC4: Petunia hybrida Vilm., Peaxi162Scf00102g01519. ASM3414082v1: Oryza sativa L. subsp. japonica, ASM3414082v1. HaNFYC4: Helianthus annuus L., LOC110935072. CiNFYC4: C. indicum L., CHR00079409-RA. StNFYC4.1/StNFYC4.2: Solanum tuberosum L., XP_006351565/XP_006351565.1. DcNFYC1: Dendrobium officinale Kimura et Migo, DOG01G001230. OsHAP5C: Oryza sativa L., Os03g0252000. GmNFYC4: Glycine max (L.) Merr., 06G16960

图2 CmNFYC4亚细胞定位及转录活性分析A：CmNFYC4亚细胞定位分析；B：CmNFYC4在酵母中转录激活活性分析；GFP：绿色荧光通道；mCherry：核定位信号；Bright field：白光通道；Merged：叠加图片。标尺=20 μm；pCL1：阳性对照；pGBKT7：阴性对照；左侧：SD/-Leu培养基；中间：SD/-His-Ade培养基；右侧：涂有20 mg/mL X-α-gal的SD/-His-Ade培养基

Fig. 2 Subcellular localization and transcriptional activity analysis of CmNFYC4A: Subcellular localization of CmNFYC4. B: Analysis of transcriptional activation activity of CmNFYC4 in yeast. GFP: Green fluorescence channel. mCherry: Nuclear localization signal. Bright field: Bright field channel. Merged: Merged image. Scale bar =20 μm; pCL1: Positive control. pGBKT7: Negative control. Left: SD/-Leu medium. Middle: SD/-His-Ade medium. Right: SD/-His-Ade medium coated with 20 mg/mL X-α-gal

图3 CmNFYC4表达模式分析A：CmNFYC4在菊花‘神马’不同组织中的表达量；B：CmNFYC4在长短日照下的表达模式分析；白框代表光照时间，黑框代表黑暗时间；试验结果以EF1α内参基因为对照，用2-∆∆Ct方法分析，数值表示平均值±SD（n=3）。图中不同字母表示差异显著，使用Tukey检验（P<0.05）

Fig. 3 Expression pattern analysis of CmNFYC4A:Expression level of CmNFYC4 in different tissues of C. morifolium ‘Jinba’. B: Analysis of the expression pattern of CmNFYC4 under long-day and short-day conditions. White boxes represent light duration, and black boxes represent dark duration. The experimental results were analyzed using the 2-∆∆Ct method with EF1α as the reference gene. Values are presented as mean±SD (n=3). Different letters above the bars indicate significant differences by Tukey test (P<0.05)

图4 CmNFYC4沉默株系鉴定及开花表型观察A：CmNFYC4沉默株系DNA水平鉴定；B：CmNFYC4在沉默株系及野生型中的表达量；C：CmNFYC4沉默株系现蕾时间统计；D：CmNFYC4沉默株系开花表型观察；NC：阴性对照；PC：阳性对照。试验结果以CmEF1α内参基因为对照，用2-∆∆Ct法分析，数值表示平均值±SD（n=3）。星号表示差异显著性，使用Tukey检验（P<0.01），标尺=5 cm，下同

Fig. 4 Identification of CmNFYC4-silenced lines and observation of flowering phenotypesA: DNA-level identification of CmNFYC4-silenced lines. B: Expression levels of CmNFYC4 in silenced lines and wild type. C: Statistics of budding time in CmNFYC4-silenced lines. D: Observation of flowering phenotypes in CmNFYC4-silenced lines. NC: Negative control. PC: Positive control. The experimental results were analyzed using the 2-∆∆Ct method with CmEF1α as the reference gene. Values are presented as mean±SD (n=3). Asterisks indicate significant differences by Tukey’s test (P < 0.01). Scale bar = 5 cm. The same below

图5 CmNFYC4瞬转过表达株系鉴定及开花表型观察A：CmNFYC4过表达株系开花表型观察；B：CmNFYC4在过表达株系及野生型中的表达量；C：CmNFYC4过表达株系现蕾时间统计

Fig. 5 Identification of CmNFYC4 transient overexpression lines and observation of flowering phenotypesA: Observation of flowering phenotypes in CmNFYC4-overexpressing lines. B: Expression levels of CmNFYC4 in overexpression lines and wild type. C: Statistics of budding time in CmNFYC4-overexpressing lines

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