The transition of meristem from vegetative to reproductive growth is an important developmental switch in the life cycle of higher plants. It is regulated by internal and external factors. These factors form a set of complex gene regulatory network in the long-term evolution and development of plants, which makes plants bloom at the best time. In recent years, a large number of studies on the floral regulation mechanism of model plants Arabidopsis and rice have found that there are six floral pathways: vernalization pathway, photoperiod pathway, thermosensitive pathway, gibberellin pathway, autonomous pathway and age pathway. In addition, FT (FLOWING LOCUS T), SOC1 (SUPPLIER OF OVER-EXPRESSION OF CONSTANTS 1) and many other key factors were isolated and identified (Blümel et al., 2015). The inhibitors of flowering also play an important role in the induction of flowering, and the silencing of the inhibitors will also accelerate the flowering. Among them, SVP gene is an important inhibitor, which is the core regulatory factor in the whole floral regulatory network. Therefore, the research on SVP gene can accelerate the improvement of flowering period of ornamental plants.

SVP gene is an important negative regulator of flowering in the STMADS11 subfamily of MADS box family. It belongs to type II MADS box protein, which has a typical MICK domain, and is highly conserved in the evolution. AGL24, another STMADS11 subfamily gene, is highly similar to SVP, but their functions are quite opposite. AGL24 is a flowering promoting factor (Michaels et al., 2003). SVP homologous genes are widely found in various plants, include many ornamental plants, and their functions are differentiated. Including kiwifruit (Wu et al., 2014), Prunus mume (Li et al., 2017), Phyllostachys violascens (Liu et al., 2016), Chrysanthemum morifolium (Gao et al., 2017), Pharbitis nil (Kikuchi et al., 2008), Paeonia suffruticosa (Wang et al., 2014), Narcissus tazetta (Li et al., 2015), Malus×domestica (Wang et al., 2019), Osmanthus fragrans (Zhu et al., 2019), eudicots (Liu et al., 2018), etc.

As a traditional Chinese flower, Chrysanthemum has a high ornamental value. Most chrysanthemum varieties are affected by short-light, with short florescence and relative concentration. Compared with other chrysanthemum varieties, day-neutral chrysanthemum with a longer flowering duration is not affected by photoperiod. It is an ideal plant to study the mechanism of chrysanthemum flowering (Gao et al., 2019). The floral transition of chrysanthemum is an important part of the study on the mechanism of day-neutral chrysanthemum. The results of transcriptome sequencing (NCBI No. srp109613) showed that the expression of CmSVP and its homologous genes was significantly different in different photoperiods. FT gene family is an important gene regulating flowering in day-neutral plants, while SVP gene inhibits flowering by down regulating FT expression (chaurasia et al., 2017; Chen et al., 2017). At present, there are FT, CO, LFY and other genes related to flowering stage in chrysanth- emum. These genes are all positive to regulate flowering stage and promote flower bud differentiation, but few are negative to regulate flowering stage. If we identify and analyze the key genes of negative regulation to inhibit flowering transformation, we can use CRISPR/Cas9 technology with simple operation and low cost, combined with genetic transformation technology to knock out the important negative regulation factors of chrysanthemum flowering, which is of great significance to cultivate early flowering varieties and continuous flowering varieties.

1 Results and Analysis

1.1 Cloning of SVP and AGL24 genes from Chrysanthemum morifolium

Two specific bands of about 700 bp and 500 bp were cloned from C. morifolium ‘Jinbudiao’ using RT-PCR method (Figure 1A; Figure 1B). The two sequences showed high similarity to the SVP and AGL24 gene of various plants using BLAST, and named CmSVP1 and CmAGL24 respectively. The ORF of CmSVP1 was 678 bp, encoding 225 amino acids (Figure 2A), and that of CmAGL24 was 465 bp, encoding 154 amino acids (Figure 2B). The CmSVP1 gene has the highest similarity to the SVP in Cynara cardunculus (83.16%), and 79.21% to 81.67% similarity to the SVP gene in Helianthus annuus and Lactuca sativa. The CmAGL24 gene was 77.62% similar to JOINTLESS-like gene in Cucurbita maxima. Further comparison with BlastP showed that CmAGL24 was 93.84% and 83.1 similar to the AGL24 protein in Artemisia annua and Helianthus. These results indicated that the two genes blonged to the SVP/AGL24 family.

Figure 1 PCR products of CmSVP1 and CmAGL24 Note: A: 1~4: CmSVP1gene; B: 1~6: CmAGL24 gene; M: DL2000 DNA marker

Figure 2 The sequences and deduced amino acid sequences of CmSVP1 and CmAGL24 Note: A: CmSVP1 gene; B: CmAGL24 gene

1.2 Analysis of basic physical and chemical properties of CmSVP1 and CmAGL24

The physical and chemical properties of the CmSVP1 and CmAGL24 genes of chrysanthemum were analyzed using ExPASy online analysis software. The results showed that the relative molecular weights of CmSVP1 and CmAGL24 protein were 25.787 kD and 17.557 kD, and the theoretical isoelectric points were 9.54 and 9.26, respectively. In the analysis of polar amino acids, the total number of negatively charged amino acid residues (Asp+Glu) of CmSVP1 was 27, and the total number of positively charged amino acid residues (Arg+Lys) was 36, with more positively charged than negatively charged. CmAGL24 has a total of 21 negatively charged amino acid residues (Asp+Glu) and a total of 27 positively charged amino acid residues (Arg+Lys), with more positively charged than negatively charged. The instability coefficients of both proteins are shown as unstable proteins.

1.3 Analysis of hydrophilicity and subcellular localization of CmSVP1 and CmAGL24 protein

The subcellular localization of CmSVP1 and CmAGL24 protein was predicted online using WoLF PSORT. The results showed that the score of CmSVP1 in nucleus was 13, that of CmAGL24 in nucleus was 8, and the cytoplasmic score was 2. It can be seen that CmSVP1 and CmAGL24 proteins were located at nucleus.

The ProtScale online software was used to predict the hydrophobicity of the protein. The larger the positive value stands for the more hydrophobic, and the larger the negative value means the more hydrophilic. The average hydrophilicity coefficient (CRAVY) of CmSVP1 and CmAGL24 proteins were -0.593 and -0.335, which indicated that these proteins have a certain degree of hydrophilicity.

1.4 Analysis of secondary structure and protein transmembrane of CmSVP1 and CmAGL24

The analysis of the secondary structure of CmSVP1 and CmAGL24 protein was used SOPMA online tools. The results showed that the secondary structure of the two proteins mainly consisted of 4 forms: α-helix (Alpha helix), β-turn (β-turn), extended chain (Extended strand) and random coil (Random coil) (Table 1). Among them, the α-helix was the most structural element in the secondary structure of these two proteins, and the remaining elements were scattered in the protein.

Table 1 Bioinformation analysis of secondary structure of CmSVP1 and CmAGL24

The TMH-MM Server2.0 tool was used to predict the transmembrane region of the two proteins. The results indicated that neither protein had a transmembrane helix region, but the CmSVP1 protein acted outside the membrane and CmAGL24 acted inside the membrane.

1.5 Homology and phylogenetic analysis of CmSVP1 and CmAGL24

The homology of CmSVP1, CmAGL24 and other SVP/AGL24 proteins from different plants with high similarity were compared using Clustalw. The results showed that both proteins had a strongly conserved MADS-box region and a K-box region, which belonged to Type II type MADS-box protein (Figure 3).

Figure 3 Multiple alignment analysis of SVP and AGL24 protein Note: The box indicates the MADs-box conservative region, and the straight line indicates the K-box conservative region

The phylogenetic tree of CmSVP1, CmAGL24 and SVP/AGL24 homologous proteins of 13 other plants was constructed using MEGA6.0. The results showed that CmSVP1 was clustered with SVP proteins of many other species. CmSVP1 had the closest evolutionary relationship with TcSVP, and the close relationship between the SVP family members of herbs and woody plants was close, indicating that SVP genes were highly conservative in plants. It was worth noting that the CmSVP1 gene has a long homology with another CmSVP in chrysanthemum, and may be functionally differentiated. CmAGL24 and AaAGL24 were grouped together, and were closely related to other species AGL24 (Figure 4). Although the CmSVP1 gene has a high similarity with the CmAGL24 gene, the evolutionary tree showed that the two genes were grouped into different groups, indicating that they were far from each other with different functions.

Figure 4 The phylogenetic tree of protein system Note: AaAGL24: Artemisia annua AGL24; CmAGL24: Chrysanthemum morifolium AGL24; HaAGL24: Helianthus annuus AGL24; LsAGL24: Lactuca sativa AGL24; ZjAVP: Ziziphus jujuba AVP; PsSVP: Pisum sativum SVP; CsSVP: Crocus sativus SVP; HfSVP: Hemerocallis fulva SVP; CmSVP: Chrysanthemum morifolium SVP; PtSVP: Populus tomentosa SVP; AtSVP: Arabidopsis thaliana SVP; DlSVP: Dimocarpus longan SVP; HaSVP: Helianthus annuus SVP; TcSVP: Tanacetum cinerariifolium SVP; CmSVP1: Chrysanthemum morifolium SVP1

2 Discussion

MADs-box family genes which are divided into Type I and Type II are an important class of eukaryotic transcriptional regulators. The Type II genes are composed of four domains with different degrees of conservation: MADs-box, I, K, and C domains, so they are also known as MIKC-type MADs-box genes that have been the most widely studied in recent years (Pařenicová et al., 2003). In flowering plants, the MADs-box family genes play an important role, and the MIKC-type gene mainly regulates floral organ development and flowering time. The SVP gene is an important member of the MIKC-type gene family and has been verified to participate in flowering regulation, flower development, dormancy and other processes in various plants (Brill and Watson, 2004; Lee et al., 2007; Jaudal et al. , 2014; Yamagishi et al., 2016). SVP and AGL24 have the closest genetic relationship and the highest homology, but they have opposite roles in regulating flowering. However, not all SVP genes have a negative regulatory effect, and some also promote flowering. The SVP homologous genes in Phyllostachys violascens and soybean promote flowering (Liu Shinan, 2016; Zhang et al., 2016), and some SVP genes are not involved in the regulation of flowering time, such as ryegrass LpMADS10, alfalfa MtSVP1 (Petersen et al., 2006; Jaudal et al., 2014). Therefore, isolating SVP homologous genes play an important role in elucidating the regulation of chrysanthemum flowering.

In this study, two SVP homologous genes named CmSVP1 and CmAGL24 were isolated in chrysanthemum by RT-PCR method. The physical and chemical properties, hydrophobicity, transmembraneity, subcellular localization and protein secondary structure of CmSVP1 and CmAGL24 were analyzed using a series of bioinformatics methods. The results showed that both genes belonged to the MADs-box gene of the MIKC type and have highly conserved MADs-box and K-box domains. The same hydrophobicity, transmembrane domain, and subcellular localization of two genes also verified their homology. Phylogenetic trees further verify that CmSVP1 and CmAGL24 were grouped into different groups and may have different functions. The SVP gene participates in plant autonomous pathways, temperature-sensitive pathways, GA pathways, photoperiod pathways and vernalization pathways, and promotes flowering by inhibiting the expression of flowering promoting factors FT and SOC1 genes (Chen Jinwen et al., 2017). SVP and AGL24 genes can interact with each other, through the formation of complexes with AP1, LUG and SEU to down-regulate the expression of promoting factors, thereby inhibiting the floral transformation. Therefore, based on the basis of this research, the function and mode of action of CmSVP1 and CmAGL24 would be further clarified. It will lay a foundation for the study of the chrysanthemum flowering mechanism.

3 Materials and Methods

3.1 Materials

The experiment used the typical day-neutral C. morifolium 'Jinbudiao' (the natural flowering period lasts from May to November) as the research material. They were planted in the greenhouse of Beijing Forestry University, and when they grew to 7 true leaves, the stem tips were taken and immediately placed Quick-frozen in liquid nitrogen and stored in the refrigerator at -80℃. The RNA extraction kit was purchased from Beijing Huayueyang Biological Company, the PCR high-fidelity enzyme, cDNA synthesis kit, pEASY-Blunt vector, and EH5α were all purchased from TransGen Biotech.

3.2 Cloning of two SVP homologous genes in Chrysanthemum

Using RNA (polysaccharide polyphenol plant total RNA) extraction kit, the preserved stem tips were extracted from the plant total RNA according to the operating instructions.

The RNA was used 0.8% agarose gel electrophoresis and NanoDrop 2000 ultra-micro ultraviolet-visible spectrophotometer to detect the concentration, purity and integrity. The first strand of cDNA was synthesized using a cDNA synthesis kit and stored at -20℃ for gene cloning.

Based on the transcriptome data obtained earlier (NCBI accession number SRP109613), specific primers for CmSVP1 and CmAGL24 genes were designed (Table 2), and PCR amplification was performed according to the following system: 1 μL cDNA template, 1 μL upstream primer CmSVP1-F (CmAGL24-F) and downstream primers CmSVP1-R (CmAGL24-R) (10 μmol/L), 12.5 μL PCR MIX (high fidelity), ddH2O make up to 25 μL. The reaction program was 94℃ for 5 min, 94℃ for 30 s, 58℃ for 1 min, 72℃ for 30 s, 35 cycles, 72℃ for 10 min, and then stored at 4℃.

Table 2 The primers in this study

The gel of the target band after electrophoresis detection was recovered and purified. The recovered DNA was connected to pEASY-Blunt vector and transformed into EH5α, and 6 monoclonals were picked and sent to BGI for sequencing.

3.3 Analysis of biological information of gene sequence and protein

The sequence after sequencing was compared by Blast program on NCBI website. ORF finder was used to predict the ORF region and deduced amino acid sequence of CmSVP1 and CmAGL24 gene. The physical and chemical properties of proteins were analyzed using ExPASy online analysis software (http://web.expasy.org/protparam/). The subcellular localization of protein was predicted online using WoLF PSORT (https://www.genscript.com/ wolf-psort.html). The protein hydrophilicity and hydrophobicity prediction were used ProtScale online software (https://web.expasy.org/protscale/). Protein secondary structure analysis was used SOPMA online tools (https:// npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html). The protein transmembrane region prediction was used TMH-MM Server 2.0 tool (http://www.cbs.dtu.dk/services/TMHMM/). The SVP/AGL24 homologous protein sequences of multiple species were downloaded from GenBank (http://www.ncbi.nlm.nih.gov/), and used ClustalW to perform multi-sequence homologous alignment of proteins between different plant species. Simultaneously, the conserved sites of CmSVP1/CmAGL24 protein of chrysanthemum were analyzed. Phylogenetic trees of SVP/AGL24 proteins of different plants were constructed using MEGA6.0 software.

Authors’ contributions

Gao Yaohui is the experimental designer and the experimenter. She completed the data analysis and the writing of the first draft of the manuscript; Xiao Fengjie and Zhang Xiaomin participated in the analysis of the experimental results; Wei Guangpu and Ma Bin completed the modification of the manuscript. All authors read and agreed to the final manuscript.

Acknowledges

This research was co-funded by the Inner Mongolia Autonomous Region Higher School Science and Technology Research Project (NJZY20094) and the Inner Mongolia University of Science and Technology Innovation Fund (2019QDL-B24).

References

Blümel M., Dally N., and Jung C., 2015, Flowering time regulation in crops-what did we learn from Arabidopsis? Curr. Opin. Biotechnol., 32: 121-129

https://doi.org/10.1016/j.copbio.2014.11.023

PMid:25553537

Brill E.M., and Watson J.M., 2004, Ectopic expression of a Eucalyptus grandis SVP orthologue alters the flowering time of Arabidopsis thaliana, Funct. Plant Biol., 31(3): 217-224

https://doi.org/10.1071/FP03180

Chaurasia A.K., Patil H.B., Krishna B., Subramaniam V.R., Sane P.V., and Sane A.P., 2017, Flowering time in banana (Musa spp.), a day neutral plant, is controlled by at least three FLOWERING LOCUS T homologues, Sci. Rep., 7: 5935

https://doi.org/10.1038/s41598-017-06118-x

PMid:28724905 PMCid:PMC5517511

Chen J.W., He X.H., Luo C., Fan Y., Zhang X.J., and Yu H.X., 2017, Research progress of plants flowering suppressor homologous genes of SVPs, Fenzi Zhiwu Yuzong (Molecular Plant Breeding), 15(12): 4888-4898

Gao Y.H., Gao Y.K., Fan M., Yuan L., Wu Z.P., and Zhang Q.X., 2017, Overexpression of Chrysanthemum morifolium SVP gene delays blossoming and regulates inflorescence architecture in transgenic Arabidopsis, Canadian Journal of Plant Science, 97(6): 1130-1139

Gao Y.H., Gao Y.K., Wu Z.P., Bu X.L., Fan M., and Zhang Q.X., 2019, Characterization of TEMINAL FLOWER1 homologs CmTFL1c gene from Chrysanthemum morifolium, Plant Mol. Biol., 99(6): 587-601

https://doi.org/10.1007/s11103-019-00838-6

PMid:30762161

Jaudal M., Monash J., Zhang L., Wen J., Mysore K.S., Macknight R., and Putterill J., 2014, Overexpression of Medicago SVP genes causes floral defects and delayed flowering in Arabidopsis but only affects floral development in Medicago, J. Exp. Bot., 65(2): 429-442

https://doi.org/10.1093/jxb/ert384

PMid:24249713 PMCid:PMC3904704

Kikuchi R., Sage-Ono K., Kamada H., Handa H., and Ono M., 2008, PnMADS1, encoding an StMADS11-clade protein, acts as a repressor of flowering in Pharbitis nil, Physiol. Plant., 133(4): 786-793

https://doi.org/10.1111/j.1399-3054.2008.01104.x

PMid:18433417

Lee J.H., Park S.H., Lee J.S., and Ahn J.H., 2007, A conserved role of SHORT VEGETATIVE PHASE (SVP) in controlling flowering time of Brassica plants, Biochim. Biophys. Acta, 1769(7-8): 455-461

https://doi.org/10.1016/j.bbaexp.2007.05.001

PMid:17566572

Li X., Wu W., Zhang X., Qiu Y., Zhang W., Li R., Xu J., Sun Y., Wang Y., and Xu L., 2015, Narcissus tazetta SVP-like gene NSVP1 affects flower development in Arabidopsis, J. Plant Physiol., 173: 89-96

https://doi.org/10.1016/j.jplph.2014.08.017

PMid:25462082

Li Y., Zhou Y., Yang W., Cheng T., Wang J., and Zhang Q., 2017, Isolation and functional characterization of SVP-like genes in Prunus mume, Sci. Hortic., 215: 91-101

https://doi.org/10.1016/j.scienta.2016.12.013

Liu S.N., 2016, Cloning and functional analysis of SVP-like and SOC1-like genes from Phyllostachys violascens, Dissertation for Ph.D., Beijing Forestry University, Supervisors: Ma L.Y., and Fang W., pp.13-61

Liu X., Sun Z., Dong W., Wang Z., and Zhang L., 2018, Expansion and functional divergence of the SHORT VEGETATIVE PHASE (SVP) genes in Eudicots, Genome Biol. Evol., 10(11): 3026-3037

https://doi.org/10.1093/gbe/evy235

PMid:30364940 PMCid:PMC6251477

Michaels S.D., Ditta G., Gustafson-Brown C., Pelaz S., Yanofsky M., and Amasino R.M., 2003, AGL24 acts as a promoter of flowering in Arabidopsis and is positively regulated by vernalization, Plant J., 33(5): 867-874

https://doi.org/10.1046/j.1365-313X.2003.01671.x

PMid:12609028

Pařenicová L., Folter S.D., Keffer M., Horner D.S., Favalli C., Busscher J., Cook H.E., Ingram R.M., Kater M.M., Davies B., Angenent G.C., and Colombo L., 2003, Molecular and phylogenetic analyses of the complete mads-box transcription factor family in Arabidopsis, Plant Cell, 15(7): 1538-1551

https://doi.org/10.1105/tpc.011544

PMid:12837945 PMCid:PMC165399

Petersen K., Kolmos E., Folling M., Salchert K., Storgaard M., Jensen C.S., Didion T., and Nielsen K.K., 2006, Two MADS-box genes from perennial ryegrass are regulated by vernalization and involved in the floral transition, Physiol. Plant., 126(2): 268-278

https://doi.org/10.1111/j.1399-3054.2006.00600.x

Wang S., Xue J., Ahmadi N., Holloway P., Zhu F., Ren X., and Zhang X., 2014, Molecular characterization and expression patterns of PsSVP genes reveal distinct roles in flower bud abortion and flowering in tree peony (Paeonia suffruticosa), Canadian Journal of Plant Science, 94(7): 1181-1193

https://doi.org/10.4141/cjps2013-360

Wang S.X., Zuo X.Y., Xing L.B., Fan S., Zhang D., Han M.Y., and Zhang L.S., 2019, Cloning, expression pattern and promoter activity analysis of flowering regulatory gene SVP in apple (Malus×domestica), Yuanyi Xuebao (Acta Horticulturae Sinica), 46(8): 1445-1457

Wu R., Wang T., McGie T., Voogd C., Allan A.C., Hellens R.P., and Varkonyi-Gasic E., 2014, Overexpression of the kiwifruit SVP3 gene affects reproductive development and suppresses anthocyanin biosynthesis in petals, but has no effect on vegetative growth, dormancy, or flowering time, J. Exp. Bot., 65(17): 4985-4995

https://doi.org/10.1093/jxb/eru264

PMid:24948678 PMCid:PMC4144777

Yamagishi N., Kume K., Hikage E., Takahashi Y., Bidadi H., Wakameda K., Saitoh Y., Yoshikawa N., and Tsutsumi K., 2016, Identification and functional analysis of SVP ortholog in herbaceous perennial plant Gentiana triflora: Implication for its multifunctional roles, Plant Sci., 248: 1-7

https://doi.org/10.1016/j.plantsci.2016.04.003

PMid:27181941

Zhang H., Yan H., Zhang D., and Yu D., 2016, Ectopic expression of a soybean SVP-like gene in tobacco causes abnormal floral organs and shortens the vegetative phase, Plant Growth Regul., 80(3): 345-353

https://doi.org/10.1007/s10725-016-0173-z

Zhu Y.M., Wang Q.Q., Dong B., Zhang C., and Zhao H.B., 2019, Effect of OfSVP on flower bud differentiation in response to ambient temperature in Osmanthus fragrans, Yuanyi Xuebao (Acta Horticulturae Sinica), 46(6): 1134-1144