Isolation and functional analysis of CYC2d orthologous genes from several plants of the tribe Anthemideae
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摘要: CYC同源基因作为控制花瓣对称性形成的关键基因，在菊科头状花序中主要调控着舌状花(两侧对称)的生长发育。菊科植物中舌状花的有或无及其分子调控机制和演化过程一直备受关注。本研究从花型不同的川甘亚菊、戈壁短舌菊和神农香菊中分别同源克隆了转录因子基因CYC2d。3个序列与菊花CmCYC2d基因氨基酸序列的比对结果显示其同源性均超过90%，且均含有保守的TCP和R结构域。半定量RT-qPCR结果显示，CYC2d在地被菊品种‘毛香玉’幼嫩花序中的表达量最高，而在川甘亚菊和戈壁短舌菊中的表达量非常微弱。因此，进一步通过实时荧光定量检测了CmCYC2d在‘毛香玉’6个发育时期舌状花和管状花中的表达情况，结果表明, CmCYC2d各个时期管状花中的表达量均很低，而在相应时期舌状花中的表达均很丰富。在不同花型的杂交F1代优株中，CmCYC2d也主要在不同位置的舌状花中高表达。通过农杆菌转化重组质粒pSUPER1300-CmCYC2d-GFP在烟草表皮细胞瞬时表达，亚细胞定位结果显示其定位于表皮细胞核。分别在野生型拟南芥和tcp1突变体(SALK-022364)中过表达CmCYC2d基因的结果表明，转基因阳性株系的营养生长受到抑制，花期延迟，花瓣大小和排列均发生了变化，使原本辐射对称的花瓣呈现两侧对称的趋势。从研究结果可知, 转录因子基因CmCYC2d对菊花舌状花的发育有重要调控作用。本研究为菊科舌状花演化的分子调控机制研究奠定了基础。Abstract: As the important gene of regulating flower symmetry, cyc-like proteins have been shown to mainly regulate the identity and development of ray floret (bilaterally symmetrical) in Asteraceae. The presence or absence of ray floret in Asteraceae and its molecular regulation mechanism as well as the evolutionary process have been highly concerned. Orthologous genes of CYC2d from Ajania potaninii, Brachanthemum titovii and Chrysanthemum indicum var. aromaticum were obtained by homology-based cloning. Their sequence alignment and conserved motif analysis were performed with the amino sequence of CmCYC2d, respectively. The results showed that their homology was more than 90% and all these proteins contained the conserved TCP and R domains. Furthermore, according to the result of semi-quantitative RT-PCR assay, CYC2d was strongly expressed in the young inflorescence of the groundcover chrysanthemum 'Mao xiangyu', while slightly expressed in that of A.potaninii and B.titovii. Therefore, the transcription levels of CmCYC2d were examined in ray and disc florets of 'Mao xiangyu' at six developing stages using quantitative real-time PCR. The results indicated that it was weakly expressed in disc florets of all stages, while highly expressed in ray florets of the corresponding stages. Moreover, in three F1 progenies with various whorls of ray florets, the CmCYC2d was expressed at much higher levels in ray florets of different whorls than in disc florets. The recombinant plasmid pSUPER1300-CmCYC2d-GFP was transiently expressed into the epidermal cells of Nicotiana benthamiana by agrobacterium-mediated transformation, and subcellular localization analysis revealed that the CmCYC2d protein mainly localized into the nucleus of epidermal cells. Furthermore, CmCYC2d was overexpressed in wild type Arabidopsis and the TCP1 mutant used the floral-dip method. The results showed that the vegetative growth and the flowering time of the positive transgenic lines were repressed and postponed. Moreover, the size and arrangement of the petals seemed to be changed, making the petal arrangement showed bilateral symmetry from original radial symmetry. These results indicate that the transcription factor CmCYC2d is essential in regulating ray floret identity in chrysanthemum. Our study lays a foundation for the research of molecular mechanisms for the evolutionary process of ray floret in Asteraceae.
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Keywords:
- flower symmetry /
- Chrysanthemum /
- Anthemideae /
- CmCYC2d /
- ray floret /
- gene expression
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图 1 地被菊‘毛香玉’花序的6个生长阶段
Figure 1. Six inflorescence developing stages of the groundcover chrysanthemum 'Mao xiangyu'
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图 2 六倍体菊杂交F1代3个优株F1-1、F1-2和F1-3
Figure 2. Three F1 progenies of two hexaploid chrysanthemum: F1-1, F1-2 and F1-3
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图 3 CYC2d同源基因氨基酸序列比对
*为川甘亚菊和戈壁短舌菊与菊花CYC2d基因在TCP和R结构域有差异的位点；黑圆点表示bHLH的核定位信号区。
Figure 3. Alignment of amino acid sequence of CYC2d homologues
* indicates the different sites in TCP and R domain of CYC2d proteins among Ajania potaninii, Brachanthemum titovii and Chrysanthemum; The black circle indicates the nuclear localization signal site.
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图 4 半定量RT-PCR检测CYC2d在4种材料花芽中的表达
MXY为‘毛香玉’，Ci为神农香菊，Ap为川甘亚菊，Bt为戈壁短舌菊。
Figure 4. Expression of CYC2d in floral buds of four plants detected by semi-quantitative RT-PCR
MXY, 'Mao xiangyu'; Ci, C. indicum var. aromaticum; Ap, A. potaninii; Bt, B. titovii.
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图 5 CmCYC2d在‘毛香玉’舌状花和管状花中的时空表达模式
A:小花不同发育时期CmCYC2d基因的表达分析。B: CmCYC2d在3个F1优株不同位置小花中的表达分析，其中：RF-O为外轮舌状花；RF-I为内轮舌状花；DF-O为外轮管状花；DF-C为中心管状花。
Figure 5. Temporal and spatial expression patterns of CmCYC2d in ray and disc florets of 'Mao xiangyu'
A: expression analysis of CmCYC2d during different stages of floret development. B: expression analysis of CmCYC2d at different floret locations of three F1 progenies. Tissues are outer ray florets (RF-O), inner ray florets (RF-I), outer disc florets (DF-O) and central disc florets (DF-C).
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图 6 CmCYC2d蛋白在烟草叶片表皮细胞中的亚细胞定位分析
Figure 6. Subcellular localization of CmCYC2d in tobacco epidermal cells
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图 7 转基因拟南芥的PCR鉴定(A)及表型分析(B)
A: M，DNA Marker; 1和2分别为质粒和野生型对照; 3~10为转基因株系；B: CmCYC2d超表达影响了植株的营养、生殖生长及花瓣排列方式。标尺：1 mm。
Figure 7. PCR identification (A) and phenotype analysis (B) of CmCYC2d overexpressing Arabidopsis
A: M, DNA marker; 1 and 2 for plasmid and wild-type controls; 3-10 for transgenic lines. B: ectopic expression of CmCYC2d in Arabidopsis affecting vegetative and reproductive growth, as well as the arrangement of petals. Scale bars: 1 mm.
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图 8 转基因拟南芥tcp1突变体的PCR鉴定及表型分析
A：M，DNA Marker 2000; 1为转空载对照; 2~11为CmCYC2d基因超表达tcp1突变体株系。B：CmCYC2d超表达抑制了根系的生长。C：CmCYC2d超表达推迟了花期。D和E：CmCYC2d超表达和空载株系的花。
Figure 8. PCR identification and phenotype analysis of CmCYC2d overexpressing Arabidopsis of tcp1 mutant
A: M, DNA marker; 1 for transgenic plants with empty vector; 2-11 for CmCYC2d overexpression tcp1 mutant lines. B: ectopic expression of CmCYC2d affecting growth of roots in positive transgenic lines; C: ectopic expression of CmCYC2d affecting flowering. D, E: flower types of the CmCYC2d overexpression lines and empty lines.
表 1 本研究所用引物序列
Table 1 Primer sequences used in this study
引物名称
Primer name引物序列(5′-3′)
Primer sequence (5′-3′)用途Use CYC2d -F1 ATGTTTTCCTCGAACCCTTTTCAT 同源克隆及半定量
RT-PCRHomology-based cloning and semi-quantitative RT-PCRCYC2d-R1 CTAGTGTAAATTTAGGAAACTTGTGTAC CnActin-F1 CACCTCTAAATCCTAAGGCTAACAG 半定量
RT-PCRSemi-quantitative RT-PCRCnActin-R1 GAACAATGGATGGGCCAGACTC CnActin-F2 CTGACAGGATGAGCAAGGAAATCAC 荧光定量
PCRQuantitative RT-PCRCnActin-R2 GAACAATGGATGGGCCAGACTC CmCYC2d-F1 TCCTCGAACCCTTTTCATCAACAG CmCYC2d-R1 GCTGCCTGTCCAAAATATTGCTGT CmCYC2d-F2 CCAAATCGACTCTAGAATGTTTTCCTCGAACCCTTTTC 亚细胞定位载体构建
Construction of pSUPER1300-CmCYC2d-GFPCmCYC2d-R2 TACCGGATCCACTAGTGTAGTGTAAATTTAGGAAACTTGTGTAC CmCYC2d-F3 GGACTCTTGACCATGGCTATGTTTTCCTCGAACCCTTTTC 转基因载体构建
Construction of pCAMBIA1304-CmCYC2dCmCYC2d-R3 CTTCTCCTTTACTAGTGTAGTGTAAATTTAGGAAACTTGTGTAC p1304-F1 ACACGGGGGACTCTTGAC 转基因植株鉴定
PCR identification of transgenic insertionp1304-R1 CAACAAGAATTGGGACAACTC p1300-F1 TCATAACCAATCTCGATACACCA p1300-R1 CTGAACTTGTGGCCGTTTACG 注：下划线处为酶切位点。Note：underlines are enzyme recognition sites. 
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