| High yield is one of the many advantages of winter rapeseed(Brassica napus L.),but it is difficult to adapt to the harsh climate in northern China in winter and over the conditions because of its poor cold resistance.There are great differences in cold resistance and overwintering of winter rapeseed(Brassica napus L.),these differences are not only manifested in morphology,but also in physiology,biochemistry and temperature sensitivity,Therefore,it has important practical significance to study the adaptability of winter rapeseed(Brassica napus L.)with different cold resistance to low temperature environment.In this study,13 winter rapeseed(Brassica napus L.)with different cold resistance were used as experimental materials.The identification index of cold resistance and DNA methylation level and pattern after low temperature stress were detected by physiological and biochemical routine detection method and methylation sensitive amplification polymorphism(MSAP).At the same time,LOC111212830 and At4g02000-like were cloned and analyzed by homologous gene cloning and real-time fluorescence quantitative PCR technique.We got the following results:1.After low temperature treatment,the activities of SOD,POD and CAT of different winter rapeseed(Brassica napus L.)with different cold resistance were increased,and the increase range of varieties with strong cold-resistant was obvious than that of varieties with weak cold-resistant,and the activities of SOD,POD and CAT increased in the weak cold-resistant varieties,which were 28.96-71.49 U/g,27.54-77.46 U/g and 11.72-16.48 U/g,the strong cold-resistant varieties were between 67.87-129.11 U/g,86.54-131.57 U/g and 22.19-43.88 U/g.2.After low temperature treatment,the contents of MDA,SP and Pro in winter rapeseed(Brassica napus L.)with different cold resistance increased,and the increase of SP and Pro in varieties with strong cold-resistant were significantly higher than those in varieties with weak cold-resistant,however,the increase of MDA was significantly lower than that of weak cold-resistant varieties,and the increase of MDA,SP and Pro of weak cold-resistant varieties varieties were significantly lower than that of weak cold-resistant varieties.In the range of 3.22-10.47 ?mol/g,3.00-7.96 mg/g,1.63-124.73 ?g/g,the varieties with strong cold-resistant,0.5-3.22 ?mol/g,9.90-21.43 mg/g,113.76-271.58 ?g/g,respectively.3.There were significant differences in DNA methylation levels among different cold-resistant winter rapeseed(Brassica napus L.).Under normal temperature treatment,the methylation level of weak cold-resistant varieties was between 19.05% and 55.56%,and the methylation level of strong cold-resistant varieties was between 20.29% and 59.57%;Under low temperature treatment,the methylation level of weak cold-resistant varieties was 45.33%-73.68%,and the methylation level of strong cold-resistant varieties was 18.18%-56.63%;Compared with normal temperature treatment,the methylation levels of 14 Meiqieshi 07,14 Meiqieshi 16,14 Meiqieshi 20,14 Meiqieshi 3 and Meiqieshi 38 increased by 25.53%,18.13%,26.29%,17.98% and 1.27%,and the methylation levels of 14NS54-1,15TS306,14NS52-3,15TS309,14NS54-7,15TS312,TSG10(Qinchuan)and TSG10(Tianshui)decreased by 0.19%,22.88%,15.77%,7.36%,2.95%,0.36%,5.32% and 2.11%.4.After low temperature treatment,the demethylation patterns were dominant in strong cold-resistant varieties such as 15TS306,14NS52-3,15TS309,14NS54-7 and 15TS312,however,the methylation patterns were dominant in weak cold-resistant varieties such as 14 Meiqieshi 07,14 Meiqieshi 16,14 Meiqieshi 20,14 Meiqieshi 3 and Meiqieshi 38.5.22 DNA methylation specific fragments selected from 13 winter rapeseed(Brassica napus L.)with different cold resistance were cloned,sequenced and compared by E86Msp32 and E1Msp36 primers,among them,16 fragments were sequence have high similarity with known and assumed functional enzymes and proteins,the LOC111212830 and At4g02000-like of Brassica napus with high similarity and unknown function were cloned and analyzed,the results showed that the length of LOC111212830 open reading frame was 1107 bp,the isoelectric point was 8.88,the relative molecular weight was 43.6KD,the instability index was 51.87,and the average hydrophilic index was-0.281;The open reading frame of At4g02000-like was 1020 bp,the isoelectric point was 9.20,the relative molecular weight was 39.9k D,the instability index was 47.60,and the average hydrophilic index was-0.437;The homology of LOC111212830 in 6 winter rapeseed(Brassica napus L.)varieties with different cold resistance(14 Meiqieshi 07,Meiqieshi 38,15TS306,15TS309,15TS312,TSG10(Qinchuan))was 97.76%;The homology of At4g02000-like was 97.17%.6.After low temperature treatment,there were significant differences in the expression of LOC111212830 and At4g02000-like in 6 winter rape varieties,such as 14 Meiqieshi 07,Meiqieshi 38,15TS306,15TS309,15TS312 and TSG10(Qinchuan),the expression of LOC111212830 conserved sequence was down-regulated in the varieties with weak cold-resistant,and up-regulated in the varieties with strong cold-resistant,At the same time,the expression of LOC111212830 specific sequence was also up-regulated in the varieties with strong cold-resistant.but the expression level was different in the varieties with weak cold resistance;The conserved sequence of At4g02000-like was down-regulated in weak cold-resistant varieties and up-regulated in strong cold-resistant varieties,The specific sequence of At4g02000-like was up-regulated in weak cold-resistant and strong cold-resistant varieties.In conclusion,under the stress of low temperature,the content of the antioxidant enzyme activity and osmotic regulators of the varieties with stronger cold resistance were significantly higher than the varieties with relatively lower cold resistance,but the content of MDA was significantly lower than the varieties with weak cold resistance;The methylated pattern and demethylated pattern coexist in the genome DNA of winter rapeseed(Brassica napus L.),in which the methylated level of the varieties with lower cold resistance rises,and methylated pattern dominates;when the methylated level of the varieties whit stronger cold resistance reduces,the demethylated pattern dominates;The relative expression of LOC111212830 and At4g02000-like in winter rapeseed(Brassica napus L.)with different cold resistance was related to the methylation pattern of the tested materials. |
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