| Low temperature is one of the important factors that limit plant growth, development and distribution. Global annual crop losses due to the low temperature damage are up to hundreds of billions of dollars. Therefore, the regulatory mechanism of plant cold tolerance and cold tolerance breeding research has very urgent demand of reality. As an important oil crops widely grown in the global scope, rapeseed showed strong tolerance to low temperature environment due to many effective cold resistance genes. In this study, we used the cold tolerant winter rapeseed variety L9 as an experiment material. Five sampling points of 0 h, 2 h, 6 h, 12 h and 24 h were determined under the condition of 4 ℃. The response mechanism of low temperature stress was investigatd from the level of RNA and protein by analysis of the physiological and biochemical indicators, cytological observation, transcriptome and proteome. The results were summarized as follows: 1. Physiological and biochemical varietions when rapseed was shortly under cold stressThere was a slight change in the leaves morphology in the early stage under low temperature stress. Further studies on the physiological and biochemical showed that the content of proline, soluble sugar, leaves conductivity, total chlorophyll concentration and the contents of chlorophyll a and b were changed with the different treatments. Under the low temperature condition, the chloroplast structure of the cold tolerant winter rapeseed line L9 was diffused gradually and its mitochondria number was increased significantly, and these cytological indexes might provide an identified reference for cold tolerance. 2. Transcriptome profiling induced by low temperature in Brassica napusThrough high-throughput sequencing and bioinformatics analysis, we assembled 71,280 to 90,041 unigenes. Among these unigenes, we obtained 31,789 to 38,201 distinct clusters and 39,491 to 51,840 distinct singletons with an average read length of 492 bp. These unigenes could be classified into 25 COG classifications. Comparisons of different treatments indicated that most differential genes(8499 up-regulated genes and 10142 down regulated genes) were specifically expressed in 12 hours after cold stress, followed by 24 hours(17824 genes), 6 hours(7901genes) and 2 hours(3198genes). The differential genes were clustered 13 groups, and divided into three broad categories according to the expression trends. After functional analysis, there were a large number of unknown genes, such as biological regulation, immune system process, metabolic process and response to stimulus, and remarkable enrichment in the pathways of circadian rhythm-plant, plant-pathogen interaction, plant hormone signal transduction and biosynthesis of secondary metabolites. 3. The dynamic control of AP2/ERF transcription factors response to low temperatureThere were 132 AP2/ERF genes identified by transcriptome sequencing of rapeseed leaves from sampling points 0, 2, 6, 12, and 24 h of low(4 °C) temperature stress. The genes were classified into 4 subfamilies(AP2, DREB, ERF, and RAV) and 13 subgroups, among which the DREB subfamily and ERF subfamily contained 114 genes, no genes were assigned to soloist or DREB A3 subgroups. One-hundred eighteen genes were located on chromosomes A1 to C9. GO functional analysis and promoter sequence analysis revealed that these genes were involved in many molecular pathways that might enhance cold tolerance in plants, such as the low-temperature responsiveness, methyl jasmonate, abscisic acid, and ethylene-responsiveness pathways. Their expression patterns revealed dynamic control at different times following initiation of cold stress; the RAV and DREB subfamilies were expressed at the early stage of cold stress, whereas the AP2 subfamily was expressed later. Quantitative PCR analyses of 13 cold-induced AP2/ERF TFs confirmed the accuracy of above results. 4. Proteomics profiling induced by low temperature in Brassica napusUsing iTRAQ technology, 3148 proteins, peptides length mainly between 8 and 13 amino acids, were indentified in the leaves which were induced with cold stress. These proteins performed to 53 GO terms and 123 pathways. Comparative analysis found, most differential proteins(320 up-regulated and 252 down regulated among 572 proteins) were specifically expressed in 24 hours after cold treatment. Pathway analysis showed the proteins were remarkable enriched in photosynthesis-antenna proteins and carotenoid biosynthesis in 2 hours after cold stress. The remarkable enrichment pathways in 6 hours after cold stress were lysine biosynthesis, photosynthesis-antenna proteins, beta-Alanine metabolism and Histidine metabolism. Photosynthesis-antenna proteins, photosynthesis, phagosome and Glyoxylate and dicarboxylate metabolism enriched in 12 hours after the treatments. There only were three pathways enriched in 24 hours which were photosynthesis-antenna proteins, Glycerophospholipid metabolism and plant-pathogen interaction. 5. Correlation analysis between transcriptomes and proteomics under cold stress in Brassic napusThe correlation analysis between proteomics and transcriptomes were conducted. There were 177 genes specifically expressed the same trend in all treatments, and annotated to 40 GO-term express a dynamic change. Such as catalytic activity and binding in molecular functions, metabolic process, cellular process and response to stimulus in biological processes. These correlated genes enriched in biosynthesis of secondary metabolites, plant hormone signal transduction and plant-pathogen interaction. There were 135 genes specifically expressed the opposite trend in all treatments, and annotated to 37 GO-term had the same functions with the same trend. |
