Analysis Of Gene Expression Profiles Of White Clover In Response To Low Temperature Stress

Trifolium repens L.is an important leguminous forage grass that is widely distributed in cold regions.Low temperature stress is a major environmental factor limiting its growth and production,and little is known about its cold tolerance at the physiological and molecular levels.In this study,the physiological changes of white clover under low temperature stress were analyzed by using 4 ℃ low temperature treatment at different times of stress,followed by transcriptome sequencing to systematically analyze the molecular mechanism of white clover under low temperature stress and to verify the functions of key genes for cold resistance.This study provides a theoretical basis for revealing the physiological and molecular regulatory mechanisms of the low temperature response of white clover,and lays the foundation for promoting the selection and breeding of cold-resistant varieties of white clover.The main findings are as follows.1.Physiological and biochemical analysis of white clover in response to low temperature stressThe low temperature stress treatment of white clover seedlings showed that CAT enzyme activity increased and then decreased with the prolongation of the stress time;chlorophyll content showed a gradual decrease;POD and relative conductivity both increased with the prolongation of the stress time and reached the maximum value at 72 h.The overall difference in relative conductivity was significant.The relative conductivity can be used as a reference indicator for the response of white clover to low temperature stress,and its change trend and magnitude can reflect the resistance of white clover to low temperature stress to a certain extent.2.Transcriptome analysis of white clover in response to low temperature stressUsing Illumina Hi Seq high-throughput transcriptome sequencing technology,5273 differentially expressed genes were identified after sequence splicing,gene function annotation and differential expression analysis.GO enrichment analysis revealed that differentially expressed genes were mainly enriched to oxidoreductase activity,transcriptional regulatory activity and metal ion binding processes.Further analysis revealed that MYB,AP2/ERF,Further analysis revealed that transcription factors such as MYB,AP2/ERF,C2H2 and b HLH played key roles in the process of low temperature stress in white clover.3.Analysis of the gene co-expression network of white clover in response to low temperature stressThirty-four co-expression modules were identified using WGCNA,of which 10 were specific to white clover.The results of GO annotation analysis showed that the darkmagenta module was significantly enriched to protein kinase activity,DNA-binding transcription factor activity and other metabolic processes,and then the co-expression network was constructed for the genes in this module,and the core genes that might be closely related to low temperature stress were screened,including CDPK,WRKY,AP2/ERF and RLK were the main genes.4.Identification and expression of CDPK in white clover leaves in response to low temperature stressEight differentially co-expressed genes associated with low temperature were validated by real-time fluorescence quantitative PCR.The results showed that the expression trends of the selected differentially co-expressed genes in low temperature treated white clover leaf tissue were consistent with the transcriptome sequencing results,verifying the accuracy and reliability of the transcriptome sequencing results.