Isolation And Functional Verification Of CYC-like Genes In Cymbidium Faberi

Cymbidium faberi Rolfe belongs to Cymbidium,Orchidaceae,which is a perennial herbaceous plant.It is native to China,also known as nine sub orchid and Chillon etc and has a long history of cultivation in China.The plant presents high ornamental and economic value because of its graceful aroma and elegant flowers.Due to the poor growth,low propagation coefficient and the long breeding cycle of natural selection,cross breeding and mutation breeding,cymbidium production can not meet the market demands.Gene engineering technique can shorten the breeding cycle and overcome the cross incompatibility barriers and therefore become a new breeding method for Cymbidium faberi.Up to date,researches on the breeding of Cymbidium faberi through gene engineering are rarely reported.Here high throughout RNA-Seq technology was used to compare the transcriptomes of vegetative and flower buds from Cymbidium faberi to understand the genes and gene networks that regulate flower development in Cymbidium faberi.We found that four TCP family CYC-like genes showed different expression levels between the vegetative and flower buds.The four CYC-like genes in Cymbidium faberi(CfCYCs)were isolated and then their expression pattern was explored.Further,the 35S:CfCYC1 was transformed into Torenia fournieri and the characteristics.of transgenic plants were analyzed to evaluate the genes' function.Our study will provide a theoretical basis for the study on floral pattern in Cymbidi um faberi.The main results are as follows:1.Through RNA-Seq sequencing,172,959 unigenes with an average length of 698bp were obtained from the flower and vegetative buds of Cymbidium faberi.These unigenes were subjected to BLASTX searches(E-value?1.0e-5)against public protein databases.As a result,a total of 65,577(37.91%)unigenes were predicted to be coding sequence.Furthermore,the 65,577 unigenes were divided into 49 functional groups by GO classification,24 clusters by COG classification,and 275 metabolic pathways by KEGG,respectively.Meanwhile,13,484 differentially expressed genes(DEGs)were identified in the two libraries using the FPKM method,of which 5,801 genes were up-regulated and 7,683 were down-regulated in the flower buds(FDR<0.05).Flowering,flower symmetry and floral organ development related genes were identified in the DEGs,and the expression levels of some of such genes in flower buds and vegetative buds were significant difference.Finally,the expression pattern of ten flower development related genes were verified by qRT-PCR,indicating that the RNA-Seq data was highly reliable.2.Total RNA of flower buds from Cymbidium faberi was extracted using the CTAB method.Based on above transcriptome sequencing and the genome database of Phalaenopsis equestris,four CYC-like TCP family genes were identified and isolated using RT-PCR and RACE techniques.Further the sequences of the four genes were analyzed.Our results suggested that the full-length of CfCYC1 gene was 1066 bp,with an open reading frame(ORF)of 927 bp,encoding 308 amino acids.The ORF of CfCYC2,CfCYC3 and CfCYC4 respectively was 975 bp,1005 bp and 870 bp,respectively encoding 324,334 and 289 amino acids.And multiple sequence alignment on TCP amino acid sequences from various species showed that CfCYCI,CfCYC2,CfCYC3,CfCYC4 had the conserved TCP-domain and R-domain,and were highly homologous to the CYC-like genes in Antirrhinum and Lotus japonicus.Phylogenetic analysis indicated that all these four CfCYCs proteins belonged to the CYC/TB1 subfamily class.3.qRT-PCR was performed to analyze the expression patterns of the four CYC/TBl-like genes.Our results indicated that the four genes expressed in floral organs and other tissues in Cymbidium faberi.Among the four genes,CfCYC1 in the flower bud,while CfCYC2,CfCYC3 and CfCYC4 in the vegetative buds showed the highest expression.Besides,the double enzyme digestion was performed to digest the original plasmids of pTG19-CfCYC1,pTG19-CfCYC2,pTG19-CfCYC3,pTG1 9-CfCYC4,the expression vector of pCAMBIA1302,and the four CfCYCs genes,conducting double restrict endonucleases ends of Nco I and Spe I,Bgl ? and Spe I,Bgl ? and Spe I,Nco I and Spe I.Then the tartget genes were cloned into pCAMBIA1302 and the recombinants' sequences were validated respectively by enzyme digestion and DNA sequencing.Thus the overexpression vector pCAMBIA1302-CfCYC1,pCAMBIA1302-CfCYC2,pCAMBIA1302-CfCYC3 and pCAMBIA1302-CfCYC4 were successfully constructed.4.The CfCYCl gene was transformed into Torenia fournieri(wild type)through Agrobacterium mediated method.Further,transgenic plants were confirmed by PCR.We found that CfCYC1 effectively affected the flower development in Torenia fournieri after morphology analysis on wild type and transgenic plants...