Transcriptome Analysis And Functional Identification Of SWEET In Daylily Under Low Temperature Stress

Daylily(Hemerocallis spp.)is a well-known perennial flower,widely used in landscaping and other fields.During the growth of daylily,it often encounters various abiotic stresses,such as low temperature stress.Researches on the molecular mechanism of the response to low temperature of daylily will provide some theoretical supports for developing cold-resistant daylily germplasm and facilitate molecular breeding.In this study,the related genes that might be involved in the response to cold stress of daylily were identified through transcriptome sequencing and bioinformatics analyses,and the members of the Sugar Will Eventually be Exported transporters(SWEET)gene family were identified in the genome-wide level,and the response of HfSWEET17 gene to low temperature stress was verified by transgenic tests.The main results of this study were as follows.1.The leaves of daylily were sampled for transcriptome sequencing after low temperature treatment.Compared with the control,2457 differentially expressed genes(DEGs)were screened after low temperature treatment of daylily,including 1253 up-regulated genes and 1204 down-regulated genes.These DEGs were mainly concentrated in 49 GO processes including catalytic activities,cellular processes and metabolic processes,and 42 KEGG metabolic pathways including biosynthesis of secondary metabolites,metabolic pathways and plant hormone signal transduction.These indicated that low temperature stress had a great effect on the physiological and metabolic pathways of daylily.The expression patterns of DEGs involved in plant hormone signal transduction and soluble sugar synthesis pathways changed to varying degrees.Among them,the expression of GH3.10 gene was 13.623 times higher than that of the control,and the expression of CWINV4 gene was 28.114 times higher than that of the control.The network regulation analysis of 29 DEGs shared by the three low temperature treatment groups showed that genes such as ABCF5,OFPs and SWEETs may occupy important positions in network regulation.According to these results,it was speculated that these genes might play important roles in the cold response of daylily.2.Sugar transporter SWEET family genes are responsible for the transport of sugar across membranes and involved in various stress response.In this study,members of the daylily SWEET gene family were identified in the genome-wide level.Bioinformatics analyses showed that 19 HfSWEETs could be divided into four clades,and the conserved motifs and domains of members of each clade were highly conserved.Except HfSWEET4 a,which contained six transmembrane domains,other HfSWEETs contained seven transmembrane domains.Chromosome location and gene collinearity analysis showed that 19 HfSWEETs were unevenly distributed on 11 chromosomes of daylily,and there were a large number of tandem repeats.Compared with Arabidopsis At SWEETs,daylily HfSWEETs were more closely related to rice OsSWEETs.The expression patterns of HfSWEETs were related to the clade in which the gene belongs.3.The results of subcellular localization analysis showed that the HfSWEET17-YFP fusion protein was mainly expressed in the tonoplast and plasma membrane,and no fluorescent signal was observed in the nucleus.The coding sequence of HfSWEET17 was amplified by PCR and inserted into the corresponding restriction site of the modified p CAMBIA1301 vector.The constructed binary vector was transformed into Agrobacterium GV3101 by heat shock transformation,and then introduced into tobacco by Agrobacterium-mediated system.The seeds of positive transgenic plants were collected and sown to T3 generation after characterization.The comparison results of phenotype and physiological indicators showed that the state of HfSWEET17 overexpressing plants at low temperature was significantly better than that of wild tobacco,the level of relative electrolyte leakage of transgenic plants was significantly lower than that of wild tobacco,and the activity of peroxidase was significantly higher than that of wild tobacco.These results indicated that the overexpression of HfSWEET17 enhanced the cold resistance of transgenic plants.