| To study the difference of cold resistance mechanism between winter rapeseed of Brassica rapa and Brassica napus, to provide reliable basis for cold resistant breeding of winter rapeseed in northern China.This study using different cold resistance of winter rapeseed varieties as materials, research on the morphological, physiological and biochemical level, protein expression level and expression of cold resistance gene level, the main results are as follows:1. The results showed that winter Brassica rapa is creeping growth, growth point is under the ground, accumulation of dry matter is mainly concentrated in the underground part, It is showed that the photosynthetic organic product of strong cold resistant varieties was preferentially allocated to the underground part during the vegetative growth stage, to establish a large root system to provide metabolic energy for the safe winter. After low temperature stress, the Brassica rapa stomata were still in open state, which was beneficial to the photosynthesis of plants, the increase of conductivity was lower, and the effective protection of the membrane structure, and the Brassica napus was the opposite.2.With the change of temperature, the physiological and biochemical indexes of different varieties was vary greatly, Stomatal conductance, transpiration rate and net photosynthesis rate of Brassica rapa reduced 96.17%,90.2%,85.22% than Brassica napus at low temperature of 4 ℃, Intercellular CO2 concentration of Brassica napus increased 11.59% than Brassica rapa at low temperature of-4 ℃. SOD activity of Brassica rapa increased 12.4% than Brassica napus at low temperature of-5 ℃,CAT、POD and SP activity of Brassica rapa increased 25.2%,40.2%,25.1% than Brassica napus respectively at 0 ℃, SS content of winter Brassica rapa average increased 29.5% than winter Brassica napus at 5 ℃, and MDA content of winter Brassica napus average increased 37.9% than winter Brassica rapa at low temperature of-10 ℃.3. Differential protein expression analysis of winter rapeseed leaves under low temperature stress: Brassica rapa Longyou7 appeared 8 new protein expression points in the leaves after low temperature stress treatment at 4 ℃, these 8 proteins were induced in expression under low temperature. Brassica napus Vision after low temperature stress treatment at 4 ℃, some protein spots will disappear, but only 4 new protein spots have been produced. Some of its in normal temperature proteins are all disappeared after 4 ℃ temperature treatment, it is showed that these proteins can not maintain their function at low temperature, and Complete degeneration and degradation.4. The c DNA sequence of beta-1,3-glucanase was isolated by RT-PCR. The c DNA sequence of this gene containing a 1032 bp opening reading frame（ORE）, encoded a polypeptide of 343 amino acid with molecular weight 38.102 k D and isoelectric point 6.63, and with 93.94% of amino sequence similarity to Brassica rapa subsp. Chinensis. The protein encoded by this gene was a hydrophilic protein had one signal-peptide. The prediction of the second structures indicated that the beta-1,3-glucanase was a steady protein with more a-helices. The predicted beta-1,3-glucanase contained a conserved amino acid sequence corresponding to the plant GlycoHydro17 superfamily, it is suggested that the subcellular localization in lysosomes synthesis. Real time RT-PCR and semi-quantitative result and showed that beta-1,3-glucanase was up-regulated expression in response to 4℃, and the gene expression to the peak in low temperature stress（-4℃）, However, the expression of this gene was inhibited at the lower temperature stress（-8℃）.5. q RT-PCR was used to analyze the difference in expression and expression pattern of Bn COR25（Brassica napus）, Bn ICE1（Brassica napus） and Cu/Zn-SOD（Brassica rapa） after the treatment of 4 ℃,-4 ℃,-8 ℃ with 5d and normal control. The results showed that the Bn COR25、Bn ICE1 and Cu/Zn-SOD were not activated and at the same level in winter Brassica rapa and Brassica napus at normal control; the relative expression of Cu/Zn-SOD, Bn ICE1 and Bn COR25 of winter Brassica rapa increased with the decrease of temperature, and reached to the peak at-4 ℃, while Bn COR25 reached the peak at 4 ℃ and Cu/Zn-SOD reached the peak at-4 ℃ in winter Brassica napus, the relative expression of Bn ICE1 is always lower than the normal temperature level in low temperature stress process; winter Brassica napus and Brassica rapa by gene expression were decreased at-8 ℃. In the whole temperature reduction process, always shows the relative expression of Bn COR25, Bn ICE1 and Cu/Zn-SOD in winter Brassica rapa was significantly higher than winter Brassica napus, cold resistant varieties was higher than weak cold resistance varieties. |
