Proteome And Transcriptome Analysis Of Response To Low Temperature Stress In Winter Rapeseed (Brassica Rapa L.)

Low temperature is one of the important ecological factors and the main abiotic stress factor which affecting plant growth,development and geographical distribution.Nowadays,enhancing the cold tolerance(cold resistance)of plants is the most effective and economical way to increase the yield of winter crops.Cold tolerance of plant was a complex trait regulated by a variety of metabolism,through a series of signal molecules to regulate the expression of related stress tolerance genes and proteins to adapting the adversity environment.With the successful implementation of the northward plan of winter rapeseed(Brassica rapa L.),the planting area of it was more and more extensive.As a new overwintering crop in northern China,winter rapeseed showed higher cold resistance.Therefore,to study the physiological and molecular mechanism of response to low temperature stress in winter rapeseed was very important to cold resistance breeding.In this study,two winter rapeseed varieties Longyou-7(ultra-cold-resistant)and Tianyou-2(weak-resistant)were used as materials to studied the physiological characteristics,photosynthetic characteristics,proteomics and transcriptomics of winter rape under gradient cooling stress.The results were as follows:1.Longyou-7 has stronger antioxidant and osmotic regulation capabilities and less cell damage,which were conducive to resisting low temperature adversity.The accumulation of ABA content is conducive to promoting cell dormancy and reducing material consumption.Both of which were necessary for the safe wintering of winter rapeseed.Moreover,the decrease in photosynthetic rate of winter rape was affected by stomatal limitation under chilling stress,while the decrease in photosynthetic rate was affected by non-stomatal limitation and under freezing stress.The chloroplast structure was severely damaged after freezing stress.The starch granules in the chloroplast were irregularly distributed in the cytoplasm and the number and volume of the starch granules were significantly increased.The chloroplast damage of ultra-cold-resistant materials had less than that in weak-resistant materials.2.According to the differential proteome analysis of winter rapeseed,there were 40 protein spots with a change more than 2 times in expression level were successfully identified in leaves.And 25 differentially expressed protein spots related to response to stress,of which 8 proteins were related to response to temperature stimulus.Five spots were up-regulated in ultra-cold-resistant materials,but not in weak-resistant materials after freezing stress.Root proteome analysis showed that there were 20 protein spots with a change more than 2 times in expression level were successfully identified.The result indicated that there were 5 spots induced expression in Longyou-7,and 6 spots in Tianyou-2 after 5 ? stress.There were 4 spots induced expression in Longyou-7 and 10 spots in Tianyou-2 after-5 ? stress.The successfully identified 20 protein spots were mainly involved in the processes of energy metabolism,protein metabolism,detoxification and defense,and so on.3.The analysis of RNA-seq showed that there were 939 differential genes(DEGs)were identified in the leaves,of which 619 were down-regulated and 320 were up-regulated,while in the roots 967 DEGs were identified,of which 542 were down-regulated and 425 were up-regulated.There were 23 significantly enriched pathways in the leaves and 14 significantly enriched pathways in the roots.It was mainly including metabolic pathways,secondary metabolic biosynthesis,phenylalanine biosynthesis and flavonoids biosynthesis.There were 71 and 78 TFs families were identified in the leaves and roots,respectively.That was divided into 19 categories in leaves,which plays a primary role in the response to low temperature stress.While in root,that was divided into 16 categories,including AP2/EREBP,C2H2,bHLH,MADS,MYB,and MYB related,and so on.4.Combined with RNA-seq and differential proteomics,low-temperature regulated protein BN115 gene COR15 b and ABA receptor protein gene PYL11 which involved in cold stress response(GO: 0009409)were selected,and performed bioinformatics analysis.Low-temperature regulated protein BN115 protein was the first time identified in Brassica napus under low temperature stress,and the COR15 b gene encoding this protein was cloned.Cloned obtained COR15 b gene with a length of 429 bp ORF which encoding 142 amino acids.The conservative region belongs to Chlorophyll A-B binding superfamily,with MW of 14.8 kDa and pI of 7.79,it was a stable hydrophilic protein.There was a tertiary structure of hydrophobic active center composed of amino acid residues at position 12,27,46,75.We also obtained the complete ORF of the ABA receptor protein gene PYL11 was 489 bp,which encoding 162 amino acids.There was a transmembrane domain with a MW of 17.8kda and a pI of 5.40,and it was an unstable hydrophilic protein.This enzyme protein belongs to the abscisic acid binding protein PYR/PYL/RCAR-like family,and there were ABA binding sites related to the hydrophobic ligand binding,protein interface,and protein switch of the PYR_PYL_RCAR_like conserved domain.The above results preliminarily elucidated the cold resistance adaptability mechanism of winter rapeseed,which enriched the cold resistance related protein and gene resources,and laid a foundation for the cold resistance breeding and cold resistance mechanism research of winter rapeseed.