Characterization And Functional Analysis Of Genes Related To Flowering In Non-heading Chinese C Abbage(Brassica Rapa Ssp.chinensis)

Brassica rapa crops belong to Brassica family of cruciferae,of which Non-heading Chinese Cabbage(Brassica rapa ssp.chinensis)originated in China along the Yangtze River in Taihu Lake region.With a long history of cultivation,Non-heading Chinese Cabbage is one of the most widely grown vegetable crops in China.Non-heading Chinese Cabbage has abundant genetic resources,the extensive introduction and hybridization at home and abroad have formed many new types of variation,making its genetic background more complicated.There exists a wide variation of flowering time in different Non-heading Chinese Cabbage cultivars.Wuyueman flowers in May and 49caixin flowers in January when sowed in September.The flowering time of the different Non-heading Chinese Cabbage cultivars must be adjusted to ensure their each other match.In this study,we cloned and identified three genes(BcMAF1,BcMAF2 and BcFLC2)from Non-heading Chinese Cabbage cultivar wuyueman.The expression level in different tissues and subcellular localization were analyzed.To investigate the fulction of the three genes,the transgenic Arabidopsis plants and Non-heading Chinese Cabbage silent plants were generated.The qPCR,yeast one hybrid and dual luciferase assay were used to screen the downstream genes.What's more,the vernalization-related candidate miRNAs in Non-heading Chinese Cabbage were isolated by sequencing.The results are as follows:1 We isolated BcMAF1 from Non-heading Chinese Cabbage and functionally characterized BcMAF1 in Arabidopsis and Non-heading Chinese Cabbage.Subcellular localization and sequence analysis indicated that BcMAF1 was a nuclear protein and contained a conserved MADS-box domain.Expression analysis revealed that BcMAF1 had higher expression levels in leaves,stems,and petals,and could be induced by short-term cold conditions in Non-heading Chinese Cabbage.Overexpressing BcMAF1 in Arabidopsis showed that BcMAF1 had a negative function in regulating flowering,which was further confirmed by silencing endogenous BcMAF1 in Non-heading Chinese Cabbage.In addition,qPCR results showed that AtAP3 expression was reduced and AtMAF2 expression was induced in BcMAF1-overexpressing Arabidopsis.Meanwhile,BcAP3 transcript was up-regulated and BcMAF2 transcript was down-regulated in BcMAF1-silencing Non-heading Chinese Cabbage.Yeast one-hybrid and dual luciferase transient assays showed that BcMAF1 could bind to the promoters of BcAP3 and BcMAF2.Taken together,our results suggested that BcMAF1 could negatively regulate flowering by directly activating BcMAF2 and repressing BcAP3.2 A systematic identification and functional analysis of BcMAF2 in Non-heading Chinese Cabbage was carried out.The BcMAF2 encoded a protein contained a conserved MADS-box domain,which was localized in the nucleus.QPCR analysis revealed that BcMAF2 which highly expressed in leaves and flowers seemed to repress flowering.To investigate this hypothesis,the BcMAF2 overexpressed Arabidopsis plants were generated and all overexpressed plants showed late flowering phenotype.Subsequently,qPCR analysis indicated that the expression of AtTEM1 was activated and AtMAF2 was repressed in the transgenic plants compared to control.In addition,we also silenced endogenous BcMAF2 in Non-heading Chinese Cabbage,causing early flowering phenotype.In those silent Non-heading Chinese Cabbage plants,the expression of BcMAF2 and BcTEM1 were down-regulated.Meanwhile,the yeast one hybrid and dual luciferase assay further revealed that BcMAF2 protein could directly bind to BcTEMl promoter and activate its expression,which was not reported in Arabidopsis.Interestingly,self-inhibition was found in BcMAF2 in Non-heading Chinese Cabbage.Taken together,this work suggested that the up-regulation of BcMAF1 is required for getting enough BcTEM1 to repress flowering in Non-heading Chinese Cabbage.3 We functional described BcFLC2 in Non-heading Chinese Cabbage.Sequence comparison to Arabidopsis FLC showed that BcFLC2 also had a MADS-box domain at the N-terminal.BcFLC2 was highly expressed in leaves,roots,stems and stamens and its expression was repressed by vernalization in Non-heading Chinese Cabbage.Interestingly,BcFLC2 expression exhibited a small peak at 2 weeks of vermalization treatment,suggesting that BcFLC2 might be involved in regulating flowering under short-term cold in Non-heading Chinese Cabbage,which is different from AtFLC expression pattern.Overexpressing BcFLC2 in Arabidopsis caused late flowering,while silencing BcFLC2 in Non-heading Chinese Cabbage caused early flowering.BcFLC2 localized to the cell nucleus and functioned as a transcription factor.Yeast one hybrid analysis revealed thatBcFLC2 could bind to the promoters of BcTEMl,BcSOC1,BcSPL15 and BcMAF2.Taken together,the present results suggested that BcFLC2 played a key role in flowering regulation as a negative regulator by controlling BcTEM1,BcMAF2,BcSOC1 and BcSPL15 expression.4 208 known and 535 novel miRNAs were obtained by sequencing,of which 20 known and 66 new miRNAs were significantly differentially expressed and considered as vernalization-related miRNAs.The corresponding targets were predicted on the basic of sequence homology search.In addition,11 miRNAs and 8 targets were selected for qPCR to confirm their expression profiles.Functional annotation of targets using GO and KEGG results suggested that most targets were significantly enriched in the hormone signaling pathway.Moreover,a decreased IAA and an increased GA3 hormone were detected after veonalization,indicating that the IAA and GA3 might response to vernalization.These results indicated that vernalization regulates flowering through microRNA mechanism by affecting hormone pathway in Non-heading Chinese Cabbage.