Study On Molecular Mechanisms Of Response To Cold Stress In Winter Rapeseed(Brassica Rapa L.)

Winter rapeseed(Brassica rapa L.)is the main oilseed crop in northern China and can safely overwinter at 35(i.e.,Tianshui,China)to 48 degrees north latitude(i.e.,Altai,Heilongjiang,Raohe,and Xinjiang,China).Winter rapeseed varieties with cold-resistance genes have increased tolerance to low air temperature and produce greater seed yield than other types of rapeseed.Understanding the mechanism of cold tolerance in winter rapeseed is of paramount importance for advancing molecular breeding of winter rapeseed and cold tolerance in other crops.In this study,two winter rapeseed varieties,Longyou-7(cold-tolerant)and Lenox(cold-sensitive)were used to determine the cold-tolerance mechanism of winter rapeseed by evaluating their morphological characteristics,physiological traits,proteomics,transcriptome levels and functions of candidate cold-tolerant genes under cold stress.The main results were:1.Longyou-7 had a lower semi-lethal temperature,and greater roots than Lenox.Longyou-7 was superior to Lenox's with regard to its protective enzyme system,which can eliminate reactive oxygen species(ROS)produced by stress in time.Its free amino acids effectively maintained the balance of regulation in cells following 3,12,and 24 h of cold stress.2.GeNorm,NormFinder,BestKeeper,and RefFinder were used to synthetically analyze the internal reference genes of winter rapeseed under four abiotic stresses.The results showed that ?-actin and SAND were the most stable combinations of internal reference genes in cold-stressed leaves,?-actin and EF1 a were the most stable combinations in cold-stressed roots,F-box and SAND were the most stable combinations in high temperature-stressed leaves,and PP2 A and RPL were the most stable combinations in high temperature-stressed roots.SAND and PP2 A were the most stable combinations in NaCl-stressed leaves,RPL and UBC were the most stable combinations in NaCl-stressed roots,RPL and PP2 A were the most stable combinations in PEG-stressed leaves,and PP2 A and RPL were the most stable combinations in PEGstressed roots.The expression profile of PXG3 was used to verify the stability of ?-actin as an internal reference gene under cold stress,which laid a foundation for subsequent gene expression experiments.3.A total of 24 proteins were successfully identified in the stem-tip meristem of Longyou-7 after cold stress,and their expression abundance showed significant changes with duration of cold stress.GO functional enrichment revealed that these proteins are closely related to transcriptional regulation,ionic stabilization,photosynthesis,and ROS scavenging enzymes in low temperature response pathways of biological processes.The KEGG metabolic pathway suggests that these proteins are primarily involved in carbon metabolism,carbon fixation in photosynthetic organisms,amino acid biosynthesis,glyoxylic acid and dicarboxylic acid metabolism,and fructose and mannose metabolic pathways.Six candidate proteins related to low temperature response were screened,and basic physicochemical properties of fructokinase-1(PF),disease-related protein(PR),and S-adenosylmethionine synthase(SAM)proteins were analyzed.The results showed that the origin of the three proteins was similar to that of cruciferous plants.There were several post-translational phosphorylated serine(Ser)and threonine(Thr)residues.PF and SAM evolution was relatively conservative,with 1 and 3 Pfam domains respectively.These results provide candidate genes for screening cold-resistant genes in winter rapeseed.4.RNA-seq analysis showed that 10,251 and 10,972 DEGs were identified in Longyou-7 and Lenox,respectively,based on the standard of p-value <0.05 and log2-Fold-Change >2.A total of 462(311 up-regulated and 151 down-regulated)DEGs were identified after 3 h of cold stress,a total of 2878(1614 up-regulated and 1264 downregulated)DEGs were identified after 24 h of cold stress in winter rapeseed.GO functional analysis showed that 127(86 up-regulated and 41 down-regulated)and 179(109 up-regulated and 70 down-regulated)were identified in Longyou-7 and Lenox,respectively.The cold stress-sensitive transcription factor families in winter rapeseed includes bHLH,NAC,ERF,and MYB_related.A total of 833,567 SNPs were detected in the transcriptome,with 48,149 and 51,090 INDELs detected in Longyou-7 and Lenox,respectively.Comparing the peroxisome pathway of Chinese cabbage winter rape,31 and 26 genes were enriched in Longyou-7 and Lenox respectively,and 13 genes were expressed simultaneously in the two varieties.Most of the genes expressed more in Longyou-7 than in Lenox at three treatment times,indicating that the metabolic activity of peroxisome in Longyou-7 was stronger under cold stress.And differentially expressed genes(Temperature-Induced Lipocalin,TIL1)closely related to cold stress were screened.5.Temperature-Induced Lipocalin(TIL1)gene participates in cold response(GO:0009409)and freezing response(GO:0050826).Cloning analysis of the TIL1(Bra002674)gene in Longyou-7 showed that the core region of this gene is 414 bp in length and encodes 137 aa.It was a hydrophilic protein with a conserved Pfam domain and belonged to the family of lipid carrier protein/cytoplasmic fatty acid binding protein(Lipocalin).Identification of TIL family genes showed that it contained similar conserved motifs and had a special amino acid sequence,there were tandem repeats between genes.The multiple homeopathic elements of the promoters were involved in stress response,which was closely related to Brassica napus(L.).The tissue expression specificity of four TIL family genes showed that the expression of TIL1(Bra002674)gene in roots,stems and tissues of strong cold-tolerant materials was significantly higher than that of cold-sensitive materials under cold stress,suggesting that TIL1 gene may play an important role in response to cold stress in strong cold-tolerant winter rapeseed.These findings reveal the molecular mechanism of cold tolerance in winter rapeseed and identified key cold-stress responsive genes and proteins,which provide rich genetic resources and theoretical basis for further investigation into their specific function and role in cold tolerance.