Chrysanthemum Floret Transcriptome Analysis And Transcription Factors Function Study Of ClCYC

Floral symmetry is an important ornamental trait of plants.It plays an important role in guiding pollination and protecting the pistil and stamens,etc.It also helps to improve plant ornamental and economic value.Chrysanthemum morifolium is native to China and is a well-known traditional flower in China.It is widely used in landscaping and is a world-renowned cut flower plant.Chrysanthemum morifolium have a unique head-shaped inflorescence,including two forms of small florets.This kind of form,the ray floret shape of the fioret is zygomorphy pattern,and the tubifrom floret is actinomorphy pattern.At present,the research on the morphology of two types of chrysanthemum flowers has begun.The research on the symmetry of flowers has mainly focused on reproductive biology and developmental biology,and relatively few new genes have been excavated.In this paper,Chrysanthemum morifolium Ramat ‘Fenditan’ was used as materials to study the transcriptome of Chrysanthemum ray floret and tubifrom floret,and two transcription factor ClCYC1 and ClCYC2 related to the regulation of flower symmetry were cloned.The genes were analyzed in different tissues by quantitative PCR The regularity of expression in the organs.The functional region of the gene is determined by subcellular localization experiments,and the function of the gene is verified by transforming Arabidopsis to observe the phenotype.Finally,the yeast two-hybrid technique is used to verify the interaction between the two proteins of ClCYC1 and ClCYC2.Conclusion as below:(1)The RNA-Seq technology was used to analyze the transcriptional response of Chrysanthemum morifolium Ramat ‘Fenditan’ ray floret and tubifrom floret.It was found that there were 8359 significantly differently expressed genes in two types of floret,of which 3005 genes were up-regulated and 5354 genes were down-regulated.GO function enrichment analysis found that more differential genes were enriched in the functional classification of biological processes,and were mainly related to cellular processes,metabolic processes and single biological processes.Pathway analysis showed that the differentially expressed genes between ray floret and tubifrom floret were mainly related to 20 metabolic pathways.The proportion of differentially expressed genes involved in phenylpropane biosynthesis,starch and sucrose metabolism,and fatty acid metabolism was the largest.(2)Using RT-PCR technology to clone two flower symmetrical regulation genes from Chrysanthemum morifolium Ramat ‘Fenditan’,named ClCYC1 and ClCYC2,the accession numbers of Gene Bank are MN746371 and MN746372,respectively.The maximum open reading frame of 900 bp encodes 272 and 299 amino acids,respectively.Physicochemical analysis showed that both genes were unstable proteins,and hydrophobicity analysis showed that both proteins were hydrophobic proteins.BLAST comparison showed that both genes had TCP domains,which are typical TCP family transcription factors.(3)Fluorescent quantitative PCR results showed that ClCYC1 and ClCYC2 genes were expressed in vegetative organs and flower organs of chrysanthemum,and the highest expression levels were found in ray floret.It is speculated that this gene may be formed in ray floret.The regulatory role played during development is higher than that of other tissues and organs.Subcellular localization experiments found that both genes only produced green fluorescence in the nucleus,indicating that the expressed proteins were localized in the nucleus,indicating that the coding products of ClCYC1 and ClCYC2 are also transcription factors and have nuclear localization functions.(4)The recombinant expression vectors p MDC85-ClCYC1 and p MDC85-ClCYC2 were constructed,and Arabidopsis plants transgenic with ClCYC1 and ClCYC2 genes were successfully obtained.The overall morphology of the transgenic Arabidopsis lines was shorter than that of the wild type,and the number of flowers was reduced The fruit pods are shortened and shrivelled,and the flower symmetry type is still the radiation symmetry type.It is speculated that the CYC gene can inhibit the development of stamens and promote the growth of petals at the later stage of flower development.(5)Yeast two-hybrid experiments showed that there is no interaction between the two proteins ClCYC1 and ClCYC2.It is speculated that the two proteins participate in different regulatory pathways,respectively.