A Study Of The Response Of Flower Symmetry And Active Ingredients To Flooding Stress In Chrysanthemum Morifolium Cv. ‘Hangju’

Chrysanthemi Flos is the dry inflorescence of the Chrysanthemum morifolium Ramat.It has the functions of scattering cold,cleaning heat andtoxin and brightening eyes,which can be mainly used to treat common cold with wind-heat syndrome,headache with vertigo,swelling and pain of eye,clouded flowery visioncarbuncle,etc.C.morifolium cv.’Hangju’belongs to one of the main cultivated lines of Chrysanthemi Flos,and its commodity value is mainly composed of medicinal value(including value for tea)and ornamental ’value(additional value).The capitulum of C.morifolium cv.’Hangju’ is mainly composed of peripheral multi-layered ray florets and a central group of disc florets,which is rich in phenylpropanoids.By comparing the origin habitat conditions of C.morifolium cv.’Hangju’and the introduction site,it was found that the origin habitat had sufficient rainfall conditions and affected the flower bud differentiation process.On the one hand,this study simulated flooding stress to analyze the transcriptome and expression profile of C、morifolium cv.’Hangju’ and dig out the information of inflorescence development and metabolism.On the other hand,by simulating soil moisture regimes to excavate related information about inflorescence morphology,active ingredient and gene expression.The main research results are as follows:(1)When flower buds begin to differentiate(Stage A),the flooding treatment(group N)was conducted for 3 days and while a control group(group F)was set,then collected the inflorescence of two groups every 15 days(StageB、C、D、E)and using the Illumina HiSeqTM 2000 platform of Huada Gene Corporation to sequence for studying the response of C.morifolium cv.’Hangju’ to flooding stress.Transcriptome sequencing produced a total of 4 805 784 720 nt data.The total number of Unigene was 101 806,the total length was 68186 645 nt,the average length was 670 nt,and the N50 reached 1 184 nt.The function annotation results show a total of 54 702 Unigene annotations,of which the number of Unigenes annotated to the NR,NT,KEGG,Swiss-Prot,COG,GO libraries are 52 480,36026,30 857,34 742,18 030,and 38 728,respectively.Gene structure analysis showed that there were 51 890 CDSs that were annotated to the protein library,7 821 CDSs were predicted,a total of 59 711;and 6 709 repeat units were detected in SSR analysis;SNP detection found a total of 314 359 base mutations,including 194 863 single base transitions and 119 496 single base transversions.The sequencing quality was good and the total amount of data met the analysis requirements.In the analysis of expression profile,the gene information in different flooding group and control group was compared with Gene Ontology and KEGG pathway,and differentially expressed genes were screened and functionally classified by group.It was found that the percentage of up-regulated differentially expressed genes in different stages of "control group vs treatment group" after flooding treatment firstly increased and then decreased with the lapse of time after dewatering treatment.Compared with the control group,the level of metabolism and secondary metabolism of the treatment group was significantly increased,and the biosynthetic activity of flavonoids in C.morifolium cv.’Hangju’ was more active after flooding;20 related sequences were screened for the key genes regulating the flower symmetry(which belong to four categories of CYCLOIDEA,DIVARICATA,RADIALIS,DICHOTOMA Gene),after drawing the heat map,it was found that the response of each gene to the flooding treatment was different.At the same time,multiple annotations were found to the "Circadian rhythm-plant "pathway.Among them,CL1419.Contig3 is a differentially expressed gene which was significantly up-regulated in the treatment group of StageD.And Unigene 26859 annotated to the " Plant hormone signal transduction "pathway.(2)The key gene for flower symmetry of CYC2c in C.morifolium cv.’Hangju’ was cloned by RT-PCR and a series of bioinformatics analysis was performed.The results show that the gene is 900 bp and encodes 299 amino acids.The molecular weight of the obtained CYC2c-encoded protein was predicted to be about 33.74 kDa,and the theoretical isoelectric point(pI)was 8.40.The secondary structure predicted by PHYRE2 showed that the disordered structure of the encoded protein accounted for 73%,the alpha helix accounted for 26%,and the beta sheet accounted for 2%.The signal peptide prediction results showed that there was no signal peptide of CYC2c protein in C.morifolium cv.’Hangju’,and the threedimensional structure model of the target protein was obtained by homology modeling method.Multiple sequence alignment results showed that the gene has a typical TCP domain which was very conserved.The phylogenetic tree construction showed that the orthologous gene with the highest similarity to CYC2c of C.morifolium cv.’Hangju’ was CmCYC1(ALP75557.1),and CYC2c was relatively conserved in Asteraceae plants with the small degree of variation.(3)In order to study the differences in inflorescence morphology,active ingredients and gene expression of C.morifolium cv.’Hangju’ under different soil moisture regimes,we set three groups of soil moisture regimes corresponding to 3 5%～40%(S1),65%～70%(S2),95%～100%(S3)of soil water holding capacity.After 62 days of treatment,samples of ray florets,disc florets and whole flowers were collected for determination of various indicators.The results of inflorescence morphology measurement showed that with the increase of soil moisture regimes,the inflorescence morphology of C.morifolium cv.’Hangju’ showed:the inflorescence diameter became smaller,the number of ray florets increased significantly,the disc florets were severely affected,and the flowering period of S3 group was significantly delayed and more malformed flowers were observed;the content of chlorogenic acid,luteolin and 3,5-O-dicaffeoylquinic acid all increased and compared with ray florets,the growth trend of various index components in disc florets was more significant and appeared to be overtaking;the gene expression measurement results showed that CYC2c gene is mainly expressed in ray florets of C.morifolium cv.’Hangju’,but weak in disc florets.At the same time,the gene expression of CYC2c in S3 group was up-regulated both in two floret samples.It was also found that the expression of FLS gene in ray florets of each treatment group had the largest difference and there were large differences in the response trends of floret samples to soil moisture regimes,and it was speculated that it may be a key gene in response to changes in soil moisture regimes and and linked to the flavonoid synthesispathway of C.morifolium cv.’Hangju’.