Research Of Conserved And Diverse Functions Of Flowering Related Genes FT/TFL1 And SOC1 In Rosacese And Petunia

The research of plant flowering mechanism has been the important and hot spots in the botany field.Currently,in the species with the different flowering habits,especially the woody plants,the comparison and research about the regulatory factors and its mechanism of the flowering regulation network is insufficient.Meanwhile,the deep research about the genes involved in flowering time control from a perspective of plant family or genus is also very rarely.As an important ornamental plant,petunia contains wide varieties and flower color.Meanwhile,it becomes one of model plants because of the characteristics of being easy for biology research.As a horticultural plant,Rosaceae family is the one of families with the most economic and ornarmental values,and contains wild variety of species and cultivars.It includes apple,pear,peach and strawberry with the high economic value fruits;also include Rosa rugosa and Rose chinensis with the high ornarmental value flowers.Rosaceae plants have a variety of growing habits,some are herbs,shrubs,even treens,and some flower in spring,summer or cold winter,and some flower once in a yearly cycle,some bloom several times or perpetually,and all these provide many plentiful materials to study the flowering regulation mechanism in woody plants.In order to understand the different flowering habits among Rosacead plants further,reveal the molecular mechanism behind these traits,and lay the theoretical foundation for traits improvement using genetic engineering,a lot of studies have been performed.FT/TFL1 and SOC1 homologs from eight Rosaceae species and petunia hybrid were isolated and functional indentified.Based on the diverse functions of FT/TFL1 genes between different species,we performed a series of experiments including site-mutated,Protein-Protein interaction and Protein-Fat binding.Moreover,another flowering pathway integrate gene SOC1,was found to be able to control flowering time not only,also can change the flower color which is closely related to flowering time.So,some preliminary research about the molecular mechanism of this phenotype has carried on.The main results were as follows:1.FT/TFL1 homologs from petunia hybrid and eight Rosaceae species namely,Prunus mume,Rosa hybrida,Fragaria ananassa,Photinia serrulata,Pyracantha fortuneana,Spiraea cantoniensis,Prunus persica and Prunus yedoensis,were isolated using the homology-based cloning combined with genome walking mothod FPNI-PCR.We found that each of the isolated FT/TFL1 g DNA sequences contained four exons and three introns,and all c DNA sequences encoded about 175 amino acids.All these amino acids sequences showed highly similar in indentity and protein structure.Ectopic overexpressing analysis of various FT/TFL1 orthologs in tobacco plants showed that there was a diversity of function among them,and we considered that it was caused by the differences of some individual amino acid residues.2.Site-directed mutations of specific amino acids in FT/TFL1 sequences were conducted by gene splicing overlap extension PCR method(SOE-PCR).All point mutagenic sequences were introduced into overexpressing vectors,and ectopic overexpressed into tobacco and Arabidopsis plants,even into the mutant Arabidopsis for function complements analysis.Through the analysis of phenotypic observation and the expression level of genes downstream by q RT-PCR method,we preliminary think that site-directed mutations of the IYN triplet motif of exon 4 can convert FT from floral promoter into TFL1-like floral repressor.Yeast two-hybrid and Bimolecular fluorescent complementation(Bi FC)analysis were used to detect the interactions of FT/TFL1 homologous or mutated proteins with the transcription factor FD.Through the induction and purification of FT/TFL1 homologous or mutated proteins in vitro,we performed Fat-western blotting to test whether these proteins have phosphatidylcholine(PC)-bingding properties.The results showed that individual amino acid change can convert the functions of FT and TFL1,but did not affect the interaction with transcription factor FD and their lipid-binding properties.Thus,our findings reveal that the divergence of flowering time modulating by FT/TFL1 homologs is independent to interaction and binding activities.3.The transgenic tobacco plants ectopic overexpressed petunia SOC1-like genes FBP21 and FBP22,or Rosaceae SOC1 homologs demonstrated an early flowering phenotype.Under the high temperature conditions(40℃,16h/28℃,8h),the flower color of transgenic tobacco plant harboring FBP21 alone and FBP21*22 both showed obvious green tune.Through the content determination of anthocyanins,proanthocyanidins,flavonols and chlorophyll in petals,we think that the increase of chlorophyll content is the main reason for the green color in petals.Meanwhile,we observed plenty of well-developed chloroplast structure in petals of FBP21*22 transgenic tabcco plant.4.Grown under normal temperature and high temperature conditions,the petals of wild-type,transgenic tobacco plants harboring FBP21 and FBP21*22 were harvested for RNA-seq analysis,respectively.Under high temperature conditions,compared to wild-type,the expression level of many genes involved in anthocyanin synthesis pathway and chlorophyll degradation pathway were down-regulated;but many chlorophyll synthesis genes were up-regulated in FBP21*22 transgenic tobacco petals.Meanwhile,the expression level of many genes involved in the chloroplast development,chloroplast RNA transcription also changed significantly.These results indicated that petunia SOC1-like gene FBP21 and FBP22 not only can regulate flowering time,also can regulate chloroplast development and chlorophyll synthesis to change flower color.