Quantitative Proteomic Analysis And Identification Of GST Genes In Hami Melon Under Cold Stress

Hami melon(Cucumis melo var.saccharinus)is rich in nutrients and is one of the main economic crops in Xinjiang,China.Cold storage is usually used to maintain the quality of the Hami melon after harvest.However,long-term low-temperature storage will cause cold damage to the Hami melon,resulting in depression of the Hami melon skin and browning-level softening,thereby affecting the commercial value of the Hami melon.Therefore,improving the cold tolerance of Hami melon after harvest is of great significance to promote local economic development.The main research contents are as follows:(1)In order to identify the genes involved in cold stress response and reveal the resistance mechanisms,cold-tolerant “Jia Shi-310”(JS)and cold-sensitive “Golden Empress-308”(GE)were selected as the materials,stored in a chamber at 0.5 ?(± 0.5 ?)with a relative humidity of 70-80 % for 0,12,and 24 days.Phenotypic index indicated that GE suffered a severe cold injury under cold stress.i TRAQ analysis showed that a total of 5291 and 5450 proteins were identified in JS and GE,respectively.Moreover,722(12 days: 391;24 days: 331)DEPs were identified in JS under cold stress,and 807(12 days:360;24 days: 447)DEPs were identified in GE.These DEPs were mainly divided into five categories:energy and carbohydrate metabolism-related proteins,stress-related proteins,structural proteins,amino acid metabolism-related proteins,and signal transduction-related proteins.The q RT-PCR verification method was used to verify eight key resistance candidate genes of Hami melon including glutathione-S-transferase(GST),heat shock proteins(HSP),et al.The results revealed a reliability of the i TRAQ data.(2)GST is a multifunctional enzyme that can catalyze the combination of GSH and hydrophobic to form conjugates,which will reduce toxic substances.In this study,a total of 39 Hami melon GST genes were identified and named according to their position on the chromosome.Phylogenetic tree analysis showed that Hami melon GST(Cm GST)can be divided into seven categories,including Tau(GSTU),Phi(GSTF),Lambda(GSTL),Theta(GSTT),Zeta(GSTZ),and tetrachlorohydroquinone dehalogenase(TCHQD)and dehydroascorbate reductase(DHAR).The members belonging to the same sub-family had the same structures and motifs.During evolution process,Hami melon was closer with squash.Besides,Cm DHAR had a strong protein-protein interaction with other Cm GSTs.Cis-elements analysis indicated that 28(70 %)Cm GSTs contained stress-related elements,which enriched in “glutathione metabolism”pathway.Under cold stress,GST,GR activity,GSH,L-glutamic acid and gamma-glutamylcysteine contents involved in “glutathione metabolism” pathway were higher in JS.The prompt transport ability in“glutathione metabolism” pathway could protect Hami melon from the damage of osmotic stress.(3)As a type of plant-specific GST,dehydroascorbate reductase(DHAR)can use GSH as a reducing agent to catalyze the reduction of dehydroascorbic acid(DHA)to ascorbic acid(As A),thereby protecting plant tissues from active oxygen damage.We identified two DHARs named Cm DHAR1 and Cm DHAR2 in Hami melon.The results of multiple sequence alignment showed that the Cm DHAR gene has a highly conserved GST-N-terminal functional domain.Collinearity analysis indicated that Hami melon was closer with zucchini,winter squash and pumpkin during the evolution process.The 3D models of protein Cm DHAR2 mainly includeed ?-helix,random coil,extended chain and ?-turn.Under cold stress,the As A contents,DHAR enzyme activity and Cm DHAR gene expression were significantly higher in JS.GSEA enrichment analysis indicated that Cm DHAR genes participated in biological processes such as ‘structural molecular activity',‘macromolecular complexes',and ‘antioxidant activity' to respond to cold stress.