Study On The Flower Color Difference Mechanism Of Cohosh And Small Cohosh

Flower is an innovative and unique trait of angiosperms,and its appearance and diversification greatly improved reproductive efficiency and environmental adaptability of angiosperms.The floral diversity of angiosperms has long been considered to be related to pollinator selection.In order to adapt different pollinators,flowers are rich in diversity in terms of shape,size,structure,color,organ composition and arrangement.Among them,flower color is an important aspect for angiosperm flowers in attracting pollinators.The flower color of most angiosperms(except Caryophyllales)is mainly determined by flavonoid anthocyanins.Anthocyanins are a class of water-soluble compounds that are bound by anthocyanin aglycones and sugars through glycosidic bonds.Different anthocyanins can be formed with different modification groups in the structure,which results in different color of floral organs.The synthetic pathway of anthocyanins is very conserved in angiosperms,and its regulation mechanism is also relatively clear.Therefore,the genetic basis of flower color transformation can be explored by a molecular mechanism study of flower color difference between closely related species,which could provide important evidences for the mechanisms of trait evolution between closely related species.Previous studies have found that Actaea purpurea and A.japonica are closely related sister species,but the flower color difference between them is obvious,and accompanied by pollinators shifts:flowers of the former are purple,and the main pollinator is Vespa bicolor Fabricius;those of the latter and other species within the genus are white,and the main pollinators are flies.In addition,the two species are genetically well diverged.Therefore,the divergence and speciation of A.purpurea and A.japonica may be mediated by pollinators shifts.This provides a great research model to study the molecular mechanisms of pollinator mediated speciation,or the molecular mechanisms of adaptation in a broader sense.In this study,I firstly identified and measured the composition and content of anthocyanins contained in A.purpurea and A.japonica sepals,and then investigated molecular mechanisms of different sepal color by transcriptome sequencing,trying to identify the target gene.Finally,the transcriptome results were verified by quantitative PCR.The main results and conclusions are as follows:1.Identification of anthocyanin and flavonoid types in A.purpurea and A.japonica sepals were conducted by UV-Vis spectrophotometer and high performance liquid chromatography.No anthocyanins were detected in A.purpurea flower buds,and the content of quercetin is 2.56?g/g;the anthocyanin contained in the sepals of the flowering period is cyanidin-3-O-glucoside,its content is 21.59?g/g,and the content of quercetin is 1.74?g/g.In A.japonica,anthocyanins were detected neither in the flower bud stage sepals nor the flowering stage ones,and the content of quercetin in the two stages was 5.79?g/g and 2.8?g/g,respectively.In addition,the content of quercetin in the flower bud of A.japonica was significantly greater than that in A.purpurea(P<0.01).2.The Illumina HiSeqTM 2000 platform was used to sequence the transcriptome of the leaves,sepals at the flower buds stage,as well as sepals at full bloom stage of each species.Three biological replications were included for each stage.After De novo assembly,193,058 transcripts and 110,338 unigenes were obtained,with an average length of 1,074 bp and 1,080 bp,respectively.The N50 is 1,550 bp and 1,619 bp,respectively.64,184 unigenes were annotated to Nr,Swissprot,TrEMBL,COG,KOG,Pram,GO,KEGG using Blastx,and the number of genes annotated into the KEGG database was 15,458.The results of differentially expressed genes showed that the expression level of the structural genes PAL,C4H,4CL,HCT,CHS,F3H,F3'H,DFR and ANS of the anthocyanins were significantly different in the A.purpurea sepals,and the peak of the expression appeared in the flower bud stage.Whereas,in the three stages of A.japonica,the genes in the anthocyanin synthesis pathway were not differentially expressed,and the expression quantity of CHI,DFR and ANS directly involved in the regulation of the synthesis of cyanidins in leaves and full blooms was very low,just a slight expression in the buds of the flower buds.In addition,the expression level of FLS and F3 'H in the flower buds stage sepals were very high,indicating that quercetin was synthesized in large quantities at this stage,which is consistent with the results of previous phytochemical methods.In the transcription factor analysis,a total of 309 R2R3MYB transcription factors were screened,of which 36 were differentially expressed;only 2 of the 25 bHLH proteins were differentially expressed;40 of 389 WD40 proteins were differentially expressed.Through sequence alignment with other three transcription factors involved in flower color formation,phylogenetic tree construction phylogenetic relationship reconstruction,amino acid sequence structure and promoter analysis,three transcription factors:ApMYB12,ApbHLHl and ApbHLH2,that may be involved in the regulation of anthocyanin synthesis were screened in A.purpurea.In addition to CHS,all the remaining screened structural genes have promoter sequences which can interact with the three transcription factors above.On the contrary,in A.japonica,transcription factors that interact with structural genes were not screened.Based on these results,it can be found that the regulation and expression of the anthocyanin biosynthetic pathway in A.purpurea sepals is normal;while the expression of most structural genes in the anthocyanin synthesis pathway of A.japonica is low,which may be due to the low expression of upstream transcription factors.In addition,the higher content of quercetin in A.japonica sepals may also be one of the reasons for results in its white appearance.In this study,the materials and molecular mechanism of the different flower color between A.purpurea and A.japonica were studied.Results show that the reason for the difference in flower color between the two species was the different expression of transcription factors rugulating cyanoside synthesis pathway.These results indicate that transcription factors could play an important role in adaptation and speciation.At the same time,this study laid the foundation for further exploring of the molecular mechanism of pollinator-mediated speciation in the system of A.purpurea and A.japonica.