Analysis Of Differentially Expressed Genes In Post-harvest Leaves Of Cut Chrysanthemum ’Fendante’ In Response To Ethylene Treatment

Chrysanthemum(Chrysanthemum morflorium Ramat.),originated from China,is a perennial herb of the family Asteraceae.As one of the top ten chinese traditional flowers and four cut flower all over the world,it wins wide popularity,and plays an important role in modern flower industry for its various colors and diverse patterns.The flower inflorescence of chrysanthemum is of relatively long shelf life,however,its leaves always wilt before the corolla senescence,which shortens the vase life.Current research on the mechanism of the effects of ethylene on senescence of cut chrysanthemum leaves are mainly focused on the physiological level.Nevertheless,the molecular mechanism of ethylene on the regulation of post-harvest senescence of cut chrysanthemum leaves is still unclear.In this study,a new generation of Illumina sequencing platform was used to find the differentially expressed genes in response to ethylene treatment in chrysanthemum,combined with the differentially expressed miRNAs and their target genes identified by miRNA deep sequencing.Gene expression regulatory network in the leaves of the post-harvest cut chrysanthemum in response to ethylene treatment was attempted,which lays the foundation for the research on the molecular mechanism of ethylene regulating senescence of cut chrysanthemum leaves.The main findings are as follows:1.Cut chrysanthemum ’Fendante’ was used as materials.200 mg·L-1 ethephon(ETP)and 1 μL·L-1 1-methylcyclopropene(1-MCP)are pre-treated for 24 hours.The results showed that the leaves vase life of cut chrysanthemum ’Fendante’ treated with ET was shortened by 10 days compared with the control,and 1-MCP can effectively delay the senescence of leaves.ET induces endogenous ethylene production in the leaves,from 0.9 nl·g-1·-1 to 3.6 nl·g-1·h-1.The Relative water content in the ET treatment group was rapidly decreased,compared with the control;While,1-MCP treatment delayed the decrease in the relative water content.The chlorophyll content in the ET treatment group was 50.9%of control in 7 d,1-MCP treatment retained the decline of chlorophyll.The MDA content of’Fendante’ treated with ET was 1.83 times compared with the control.1-MCP treatment generally reduces the increase in MDA content.Using large-scale high-throughput sequencing platform Illumina2500,libraries were constructed after water,ET,1-MCP treatment for 3 and 24 hours for cut chrysanthemum ’Fendante’ leaf transcriptome sequencing.The sequencing results showed that a total number of 1.04 Gb clean Data was obtained,Q30 was more than 90%,and the comparison efficiency of sequencing tags on the chrysanthemum reference genome was more than 70%.It was found that 3 h ET vs CK yield 738 DEGs;24 h ET vs CK,196 DEGs;3 h ET vs 1-MCP,39 DEGs;24 h ET vs 1-MCP,2,723 DEGs.KEGG analysis illustrated that the differential expressed genes were mainly related to the pathway of photosynthesis,plant hormone synthesis and signal transduction,porphyrin and chlorophyll metabolism.Twelve selectd genes were verified by quantitative RT-PCR,which from different pathways,including plant hormone gene EBF2,CTR1,EIN2 and TIF6,aquaporin gene PIP1,ROS gene PER51,transcription factor NAC72,NAC100,ERF92,RAP2-2,bHLH63 and BHLH130.The trends of up and down regulation from qRT-PCR were consist with those frome the RNA-seq,indicating that the analysis were reliable.Selecting different ethylene response cultivar with the same 12 DEGs,the verification test showed that there was a significant correlation between the expression of the different genes and ethylene treatment.In the ethylene-sensitive varietie,the induced amplitude is greater and responds to the ethylene signal more quickly.These results indicated that these genes were important candidate genes in regulation senescence of cut chrysanthemum leaves in response to ethylene.2.For cut chrysanthemum ’Fendante’ leaf miRNA sequencing,libraries were constructed using leaves after treatment of water,ET,1-MCP for 3 and 24 hours.The results showed that 21 libraries obtained a total of 370.02 million raw data,359 million clean reads.miRNAs are mainly distributed between 21-24 nt,and most of them are 21 nt in length.Known miRNAs belong to 64 different miRNA families.41 significant differentially expressed miRNAs between different treatments were detected.We found miR164,miR166,miR172,miR319 and miR396,which were previously known miRNA in response to senescence.New miRNA in response to ethylene including miR397 and miR408.Combined with transcriptome data and targets prediction analysis,it was found 32 miRNAs regulated transcription of 36 target genes,and the targets were mainly participated in processes such as transcription regulation and signal transduction.