The Responsive Mechanism Of Chrysanthemum To Low Temperature And Functional Identification Of Chrysanthemum DgWRKY4 Gene

Chrysanthemum is widely cultivated and applied in the world because of its rich variety and color,resistance to transportation,long vase life and so on.It has extremely high economic benefits.In recent years,the production of cut chrysanthemum is seriously affected by two stress factors:low temperature and high salinity.In response to these adversity,combined with results of transcriptome sequencing and physiological indicators,we investigated the response mechanism of chrysanthemum to low temperature stress,excavated potential cold-resistant genes,and identified the salt tolerance of DgWRKY4transgenic chrysanthemum.It provides an effective gene reserve for the stress resistant molecular breeding of chrysanthemum morifolium.Using Illumina sequencing technology,a total of 86,444,237 high-quality clean reads and93,837 unigenes were generated from four libraries:（T01）controls;（T02）4℃cold acclimation（CA）for 24 h;（T03）-4℃freezing treatments for 4 h with prior CA（cold acclimation）;and（T04）-4℃freezing treatments for 4 h without prior CA.In total,7583differentially expressed genes（DEGs）of 36462 annotated unigenes were identified.The research analyzed the enrichment of metabolic pathways of chrysanthemum gene products under low temperature stress,discussed the expression level changes of genes related to protein kinases,transcription factors,signal transdution factors,and the membrane system,and excavated a large number of important cold responsive genes related to low temperature sensing and signal transduction,membrane lipid stability,reactive oxygen species（ROS）scavenging and osmoregulation.These genes can be used as candidate genes for cold-tolerant molecular breeding of cut chrysanthemum.In this study,chrysanthemum seedlings treated with CA（T03）and without CA（T04）were exposed to the same freezing condition.The physiological results showed that T04 faded faster than T03.The antioxidant enzymes（POD,CAT）activities,proline and soluble sugar contents of T03 were higher than T04;while relative electrolyte conductivity,H₂O₂ and O₂^-accumulation was lower than T04.Furthermore,trough transcriptome sequencing analysis,there were more DEGs up regulated in T03 which underwent CA than T04 without CA,and the up-regulation of some important low temperature responsive genes in T03 was greater than T04.It was proved that CA could lead to the establishment of cold resistance mechanism of chrysanthemum,and finally improve the adaptability of chrysanthemum to freezing.This study identified the tolerance of DgWRKY4 transgenic chrysanthmeum to salt stress.It was found that DgWRKY4 could be induced by NaCl.Under normal conditions,the expression level of DgWRKY4 in different tissues of wild type（WT）chrysanthemum was leaf>stem>root.Under normal conditions,all chrysanthemum showed no obvious phenotypic difference at the seedling stage.Under salt stress,leaves of WT plants were yellowed and wilted,while transgenic chrysanthemum’s remained green,and the survival rate of transgenic chrysanthemum was higher.Physiological determination showed that under salt stress,the chlorophyll,net photosynthetic rate（Pn）,stomatal conductance（Gs）and transpiration rate（Tr）of chrysanthemum were higher than that of WT,and transgenic lines had lower malondialdehyde（MDA）,H₂O₂ and O₂^-contents,higher proline and soluble protein contents and higher antioxidant enzymes（SOD,POD,CAT）activities.The qPCR technique was used to detect the expression level of DgWRKY4 downstream resistance-related genes.Under salt treatment,the transcript accumulation of DgCuZnSOD,DgCAT,and DgAPX,which encode ROS-scavenging enzymes,and DgP5CS,which functions in osmotic adjustment,was increased remarkably in overexpressed lines compared to WT.Moreover,DgWRKY4 also induced the up-regulated expression of other transcription factors.The study identified DgWRKY4 was a positive regulator of salt tolerance in chrysanthemum and it can confer higher salt tolerance to transgenic chrysanthemum.So,DgWRKY4 can serve as an excellent candidate gene for salt-tolerant plant breeding.The above studies provide effective gene reserves for breeding new varieties with low temperature and salt tolerance.