Evolution Analysis And Function Identification Of RmSVP Genes In Rosa Multiflora

DAM gene is the homologous gene of SVP,which is considered to be the key factor to regulate the dormancy or seasonal flowering of most Rosaceae plants.However,the evolutionary model of the system is not clear.In this study,we identified SVP and FLC genes from genome annotations of Rosaceae and Brassicaceae species.In addition,we used wild-type Arabidopsis and svp31 mutant Arabidopsis as materials to identify the function of RmSVP gene.The main results are listed below:1.In order to gain insights into the evolution features of its structural composition in Rosaceae(mainly perennials),we performed an extensive identification of FLC and SVP genes from Brassicaceae and Rosaceae genomes,and generally compared their copy number between these two lineages,with Vitis vinifera as out group.Our results indicated that SVP2 to SVP4 clade genes were originated from SVP1 clade and specifically expanded in different lineages of Rosaceae,and in turns suggest their functional diversification in different lineages.Furthermore,ten lineage-specific gene duplication(GD)events(GD1-10)were proposed for the expansion of SVP genes.The protein sequences of different SVP genes were highly conserved,and the cis-acting elements of promoter of SVP gene in Rosaceae could be divided into five clusters.Our results indicated that the SVP genes were significantly expanded in Rosaceae while FLC genes were suffered extensive gene loss during the evolution of Rosaceae.There are no FLC gene identified in the Rosa chinensis and Rosa multiflora,in concluding,the function of FLC gene is weakened in the Rosaceae plants.This research focuses on the function of RmSVP genes in vernalization pathway.2.In order to investigate whether RmSVP genes is the key factor of ambient temperature pathway,and whether RmSVP genes expression has any relation to temperature or not,we determined the relative expression level of all five identified RmSVP genes both in leaves and buds during the whole year growth cycles.The results showed that the expression patterns of RmSVP1/2/3/4 genes in the leaves were similar.Their expressions were significantly up-regulated when the environmental temperature first closed to subzero.The expression activity of RmSVP1/2/3/4 genes were promoted when exposed to low temperature for short time and inhibited when exposed to low temperature for longer time.The expression pattern of RmSVP1/3/4/5 genes in axillary bud were much similar.In October,the expression level of these genes was high,but subsequently begin to decrease and had been in a very low state until flowering.However,the expression pattern of RmSVP2 gene in leaves and axillary buds were the same.Short-term low temperature promoted its expression,while,long-term low temperature stably inhibited its expression.Phylogenetic analysis indicated that RmSVP2 and RcSVP2 gene have the highest homology.By comparing the CDS sequences of RmSVP2 and RcSVP2 gene through BioXM2.6,we found that the sequences between RmSVP2 and RcSVP2 gene have only two bases difference,and have only one difference of amino acid.The homology relationship between RmSVP2 and RcSVP2 gene is more than 99%.In this study,the tissue culture seedlings of Rosa multiflora and Rosa chinensis were treated with low temperature,and the expression of RmSVP2 and RcSVP2 genes were detected by qRT-PCR.The results showed that the response of RmSVP2 gene to low temperature was much more significant than RcSVP2 gene.3.In order to further explore the function of RmSVP2 gene,we cloned the full length of RmSVP2 gene,and constructed the over expression vector.The ectopic overexpression of RmSVP2 gene in Arabidopsis not only affects its flowering time,but also the floral organ development.Specifically,the chlorophyll content were increased in petals and it appeared green.The pistil development is seriously deformed,causing reduced seed setting rate.4.The results showed that RmSVP2 gene was a flowering suppressor.In order to further dissect the molecular mechanism of RmSVP2 gene in regulating flowering.This experiment focuses on the relationship between RmSVP2 gene and RmFT/SOC1 genes that are the key factors affecting flowering time.It was proved that RmSVP2 protein could interact with RmFT and RmSOC1 protein by double luciferase test and yeast two hybrid test.It is proved that RmSVP2 gene can bind the promoter of RmFT gene and inhibit the activity of promoter by promoter binding test,which may be the molecular mechanism of RmSVP2 gene affecting flowering time of plants.