Gene Cloning And Functional Analysis Of CmBBX7 In Chrysanthemum

Most of chrysanthemum cultivars are short-day plants,flowering in autumn,which originated in China.As one of the four most popular cut-flower around the world,it has highly ornamental value.Summer Chrysanthemum cultivar 'Yuuka' blooms in June to September in nature conditions,and it is popular because of the nice flower pattern,pure color,straight stems,strong resistance and tolerance.On the other hand,BBX family genes have various functions.This family members can not only response to temperature and hormone,but also to light signal in photoperiod flowering pathway.The gene expression can be changed by environment conditions and then to regulate plant growth.In this research,CmBBX7 as a BBX family member,was cloned from Chrysanthemum cultivar 'Yuuka'.To analyze the function of this gene,these methods such as structure analysis,yeast two-hybrid,BiFC,transgenic technique and quantitative RT-PCR were uesed.The results of this experiment are as follows:1.In order to select different flower bud differentiation period samples to do RNA-seq,we summarized a set of paraffin section method which was laborless and time saving.According to the data of 'Yuuka' transcriptome(SRP029991),the full-length of CL7046.Contig5_All was cloned.After sequencing and phylogenetic analysis,this gene was desingated as CmCOL9,another name CmBBX7.2.Yeast transcript activity assay showed that CmBBX7 protein have transcriptional activity,and the transcriptional activation site of CmBBX7 locates at the region between the two B-boxes and the CCT domain.On the other hand,subcellular localization assay showed the fluroscent signal of CmBBX7-GFP fusion protein was detected only in the nucleus.3.The analysis of CmBBX7 expression showed that the expression pattern of CmBBX7 showed diurnal oscillations which was consistent with that of CmCO both in LD and SD condition,but the expression level were contrast at a same time.On the other hand,qRT-PCR showed that CmBBX7 has the highest expression level in leaves and lowest in root.4.Overexpression CmBBX7 in arabidopsis plants showed that transgenic lines bloomed late.And the qRT-PCR result showed that flower promoting genes,such as API,LFY,FT,CO and SOC1,expressed less and the flower inhibiting gene FLC express more in the transgenic plants than in wild type plants.5.Yeast two-hybird experiment and Bimolecular Fluorescence Complementation(BiFC)showed that CmBBX7 protein could interact with CmCO,and the specific interaction sites of these two protein were CmBBX7-B-boxl and CmCO-B-boxes domain.The result of tobacco transient assay showed that the delayed-flowering of transgenic plants may be caused by the physical interaction leading to depletion of the active CmCO for the induction of CmFTL3 expression but not degration of the CmCO protein,and the lower transcripts of CO.