Transcriptome Analysis Of The Ethylene Response In Chimonanthus Praecox And The Clonging Of CpERF1 Gene

Wintersweet（Chimonanthus praecox）is familiar as a garden plant and woody cut-flower.On account of its unique flowering time and strong fragrance,it has a high ornamental and economic value.And its ornamental value depends mainly on the opening of the flower.In order to explore whether the ethylene affects the opening of the flower,in this paper,transcriptome of control group,ethylene treatment group and1-mcp treatment group were analzed,using RNA-seq technique,to explore the effect of ethylene on flower of Chimonanthus praecox and to clone ethylene response factor gene.It is useful to regulatethe wintersweet flowers open and improve the quality of cut flowers.The main results were as follows:1.In this study,total RNA was extracted from the flower bud stage of Chimonanthuspraecox,usinghigh-throughputIlluminasequencingplatform HiSeq4000,atotalof40,264,927（0h）,41,064,633（4h）,39,230,880（4hEth）,41,158,515（24h）,40,851,828（24h1-mcp）clean reads were obtained.Trinity software was used to splice all clean reads,and 195,349 transcriptional sequences were obtained,with an average length of 919bp.2.The functional annotation of unigenes was searched against the Nr,Swiss Prot,COG,GO,and KEGG databases.A total of 52,352（26.8%）unigenes sequences of which were annotated in the NCBI non-redundant protein database,and found the high homology with the grapes.Gene functional classification was conducted based on GO database.A total of 8924 transcript sequences were classified to cellular components,molecular function,and biological processes.For COG analyses,43391 transcript sequences were classified into 25 functional categories.We could map 2,926transcripts onto 132 pathways using the Kyoto Encyclopedia of Genes and Genomes pathway database.3.Based on transcriptome sequencing results of different treatments of Chimonanthus praecox,the differentially expressed genes of 0hvs4h,0hvs24h,0hvsEth4h and 0hvs1-mcp24h were analyzed.The significant differentially expressed genes were found to be 282,396,293 and 367,respectively（Log₂FC≥2or≤-2;FDR≤0.05）.In the difference gene of 0hvs4h,108 genes were significantly up-regulated and 174 genes were down regulated significantly,while genes of 0hvsEth4h,143 genes were significantly up-regulated and 150 genes were significantly down regulated.In the different genes of 0hvs24h,127 genes were significantly up-regulated and 168genes were down regulated significantly,while gene of 0hvs1-mcp 24h,191 genes were significantly up-regulated and 176 genes were significantly down regulated.An analysis of differentially expressed genes involved in plant hormone signal transduction pathways indicated that the differential genes related to plant hormone signal transduction pathways are significantly different.These genes are annotated by plant hormone receptors or plant hormone responsive genes,such as ARF,AUX/IAA,SAUR,CKX,ERF and FBL genes.Differentially expressed genes which were Random selection was detected by Q RT-PCR technology,indicating the accuracy and effectiveness of transcriptome sequencing.In this study,we obtained the genes related to flower development,we also got the genes related to anthocyanidin,including F3PH and NCED1 genes.4.Based on transcriptome sequencing results which carried out analysis of transcription factor data,a total of 35723 transcription factors were annotated.including 94 transcription factor families,such as C2H2,C3H,NAC,bHLH,MYB,AP2/ERF and other transcription factor families.In order to understand the changes in transcriptional level of differentially expressed genes in the transcriptional data,the differential genesannotated144,161,139 and 199 transcription factors in the0hvs4h,0hvs24h,0hvsEth 4h and 0hvs1-mcp 24h,which respectivly belong to 39,47,45 and 52 transcription factor families.5.Based on the transcriptome sequencing database which was induced by the ethylene,we obtaind the AP2/ERF transcription factor famliy member gene sequence.The full-length cDNA sequence of AP2/ERF transcription factor CpERF1 gene was obtained by cloning.The analysis shows that the cDNA sequence with a full-length of852bp contains an618bp open reading frame（ORF）which encodes 205 amino acids.The transcription factor of CpERF1 contained the AP2 DNA binding domain.The CpERF1 was a typical AP2/ERF family transcription factors in plants.Homologous phylogenetic tree showed that this ERF transcription factor was the closest relatives to AtERF1（AT3G23240.1）in Arabidopsis,they all belong to group B3,one group of the ERF subfamily transcription factors.Further analysis expression characteristics of CpERF1 gene,real-time quantitative PCR showed that CpERF1 gene expressed in all tissues of Chimonanthus,and the expression in mature leaves was the highest.During flower development,the CpERF1 gene had the highest expression levelin blooming phase.5.The plant overexpression vector pCAMBIA2301g-CpERF1 was successfully constructed.The Agrobacterium tumefaciens GV3101 vector was used to transform Arabidopsis thaliana by inflorescence infection.We obtained heterozygous nine strains of T1 generation of transgenic Arabidopsis thaliana by screening.