Cloning And Functional Analysis Of CmBBX13 And CmBBX15 In Chrysanthemum 'Yuuka'

As one of Chinese traditional famous flowers and four cut flowers over the world,Chrysanthemum(C morifolium)is loved by many people.In the market,most chrysanthemum cultivars are flowering in autumn.However,chrysanthemum 'Yuuka' is the typical summer chrysanthemum cultivar from Japan and naturally flowering from June to September.Chrysanthemum 'Yuuka' is popular because of full flower pattern,pure color and strong tolerance in the cut flower companies.Research of the molecular mechanism for flowering of 'Yuuka' will provide theoretical basis to guide production and regulate flowering time.BBX proteins belong to the family of a functional diversity,the family members are involved in regulation of growth and development and responded to abiotic stress signals.So far,other plant species has many reports involved in regulation of flowering,but there is little about research progress for chrysanthemum.In this study,CmBBX13 and CmBBX15 were isolated from chrysanthemum `Yuuka'.To assay the expression patterns of CmBBX13 and CmBBX15,the methods of qRT-PCR,yeast two-hybrid and subcellular localization were used.And then we uesed transgenic technique to obtaining genetic plants of CmBBX13 involved in regulation of flowering.The main contents and conclusions are as follows:1.Three-BBBX-fenfly were obtained from the transcriptome--library-of C.morifolium'Yuuka',and the complete cDNA sequence of three genes were cloned.According to phylogenetic analysis,we designated CmBBX13 because it has the closest relationship with AtBBX13.Another two genes were relative to AtBBX15,we designated CmBBX15.1 and CMBBX15.2 through diffrent day lengths condition.2.The expression pattern of three genes were examined using the RT-PCR method.The results showed the CmBBX13 transcript abundance was highest in the leaves and apical of stem.The CmBBX15.1 and CmBBX15.2 expression levels were the highest in leaves,the lowest in roots.The expression of CmBBX13 showed no rhythmicity under the both in long day and short day.But the expression of CmBBX15.1 was significantly increased in LD conditions,CmBBX15.2 transcript levels has rhythmicity in SD conditions.Moreover,we found that these genes responded to cold,drought and salt stress.But the signal pathways remains to be futher studied.3.The subcellular localization indicated that CmBBX13,CmBBX15.1 and CmBBX15.2 proteins were localized in the nucleus.Yeast one-hybrid assay showed that CmBBX15.1 and CmBBX15.2 proteins has transcription activity except for CmBBX13 protein,and the domain of transcriptional activation were located at region between the B-box and the CCT domain.4.We constructed the plant expression vector pDEST-35AA-SRDX-CmBBX73(with stop codon)pMDC43-CmBBX15.1,pDEST-SRDX-Cm5BBX15.1,pMDC43-CmBBX15.2,pDEST-SRDX-CmBBX15.2 and obtained Arabidopsis transgenic lines selected by resistance and qRT-PCR.We found CmBBX13 transgenic Arabidopsis plants had much more rosette leaves than the control plants with about 5-day delay in flowering under long day conditions.Moreover,The RT-PCR results showed flowering related genes such as API,SOC1,FUL,LFY,FT,FD and CO were down-regulated in Arabidopsis transgenic lines,while FLC,TOE2 and FRI in the transgenic lines were upregulated more than wild type plants.Compared with wild type plants,CmBBX15.1 and CmBBX15.2 transgenic Arabidopsis had no significant difference in flowering time.We speculated CmBBX15.1 and CmBBX15.2 had no direct influence in flowering time.The AtBBX13 mutant bbx13 showed early flowering in Arabidopsis under long-day conditions.