The Molecular Mechanism Of Chrysanthemum CmTPL1-2 Involved In The Flowering Regulation

Chrysanthemum(C.morifolium)is one of the ten famous flowers in China and one of the top four popular cut flowers across the world,provides high ornamental and economic value,which plays an important role in the floriculture industry.The high energy cost for the year-round products challenges the sustainale Chrysanthemum producing.In this study,the function of Chrysanthemum TOPLESS 1-2(CmTPL1-2)was further studied by transgenic methods using traditional Chrysanthemum 'Jinba' as material,and the downstream genes was identified via RNA-seq.The interaction protein of CmTPL1-2 was screened by yeast two-hybrid system,and the screened proteins were verified in vitro and in vivo.Taken together,the molecular mechanism of CmTPL1-2 protein complex in regulating the flowering time will be revealed through the present study.The study is expected to make a breakthrough in the molecular mechanism of strigolactone(SL)signal regulation of Chrysanthemum flowering,providing excellent genes for molecular breeding of plant flowering.The main contents and conclusions are as follows:1.Exogenous SL synthetic analogue GR24 spray treatment of Arabidopsis thaliana and Chrysanthemum 'Jinba',SL signal pathway gene TPL/TPR expression was down-regulated,plant floweringed earlier,however,the mechanism of specific flowering regulation was unclear.It is indicated that SL can inhibit the expression of TPL/TPR,and that TPL/TPR is involved in the process of SL regulation of flowering.On this basis,CmTPL1-2 gene was cloned in Chrysanthemum 'Jinba'.Tissue quantification revealed that the expression level of CmTPL1-2 gene was the highest in shoot tips and low in roots and stems.To further elucidate the function of CmTPL1-2,a mut-CmTPL1-2(N176H)mutant sequence was constructed for further study.Subcellular localization of onion epidermal cells revealed that CmTPL1-2 and mut-CmTPL1-2 were localized in the nucleus and the transcription inhibition was verified with assay.2.Transformation of CmTPL1-2 and mut-CmTPL1-2 into Arabidopsis thaliana revealed that CmTPL1-2 overexpressing lines flowering later,while mut-CmTPL1-2 overexpressing lines flowering earlier.Quantitative verification of key flowering genes FT,TSF,FUL and AP1 revealed that the relative expression levels were down-regulated in CmTPL1-2 overexpressing lines,while up-regulated in mut-CmTPL1-2 overexpressing lines.Furthermore,CmTPL1-2 and mut-CmTPL1-2 transgenic Chrysanthemum 'Jinba' still showed the corresponding phenotype of flowering phenotype.To explain this phenomenon,the dimer interaction was discovered by means of protein interaction.When the amino acid at position 176th of CmTPL1-2 is mutated from asparagine to histidine,it can interact strongly with unmutated wild-type CmTPL1-2.It is speculated that mut-CmTPL1-2(N176H)may cause loss of function of the TPL/TPR protein family,resulting in differences in flowering time of plants.By transcriptome sequencing of CmTPL1-2 transgenic Chrysanthemum,many photoperiod pathway-related genes including CO,COLs,CCA1,LHY,TOC1/PRR1,PRR7,AP2/TOEs expression were changed,gibberellin(GA)pathway-related genes including GRAS1 DELLAs,GA2ox and possibly responding SVP and other genes were significantly up-regulated.The key flowering regulatory genes FT,AGL20,AGL42,AP1/FUI and FUL/CDM41 were down-regulated.It is speculated that these differentially expressed genes are responsible for the late flowering of the CmTPL1-2 transgenic line.3.In order to clarify the molecular mechanism of Chrysanthemum CmTPL1-2 involved in flowering regulation,a yeast library before flower bud differentiation of Chrysanthemum 'Jinba' was constructed,and CmTPL1-2 was used as bait protein to screen its interaction protein.Co-transfection in yeast showed that CmTPL1-2 could interact with CmSVP,and it was found that both CmTPL1-2 and CmSVP could interact with the Cl-terminus of CmJ3,and further proved by the mutual verification method of onion BiFC and tobacco FLuCI protein.There are interactions between each of CmTPL1-2,CmSVP and CmJ3.It is speculated that CmJ3 needs to be modified in plants to function.Overexpression of CmSVP and CmJ3 in Arabidopsis thaliana revealed that CmJ3 transgenic lines flowered earlier,CmSVP transgenic lines flowered later,which laid a solid foundation for further research on the regulation of Chrysanthemum flowering at the translation level by CmTPL1-2.