Differential Proteomic Analysis Of Winter Rape (Brassica Napus L.) Under Low Temperature Stress

Winter rape(Brassica napus L.)has many advantages such as high yield and lodging resistance,but its poor cold resistance makes it a limiting factor for planting in the north.The cold resistance of plants is a complex regulatory network,involving many regulatory mechanisms.In this study,the effects of low temperature stress on winter rape varieties with different cold resistance were discussed from the differences of morphological characteristics,physiological and biochemical characteristics,photosynthetic characteristics and protein characteristics.The main results are as follows:The effects of low temperature stress on the morphology,physiological and biochemical characteristics of Brassica napus seedlings were studied.the results showed that after 24 hours of treatment at-4 ℃,the leaves of Brassica napus wilted and drooped,the leaf color deepened,and the plants grew creeping or semi-creeping.The activities of antioxidant enzymes(SOD,CAT,POD,APX),osmotic substances(SS,SP,Pro),malondialdehyde content(MDA)and relative electrical conductivity of Brassica napus with different cold resistance showed an increasing trend,but the increasing range was obviously different due to the difference of cold resistance of varieties.The increments of antioxidant enzyme activity and osmotic adjustment substance content from large to small are as follows: strong cold-resistant varieties,weak cold-resistant varieties and cold-sensitive varieties.However,the increase of malondialdehyde and relative conductivity is just the opposite.The results showed that the varieties with strong cold resistance could maintain the integrity of plasma membrane by increasing the activity of enzymes and the content of osmotic regulators after low temperature stress.At room temperature,the stomata of Brassica napus-type winter rape were open,the chloroplast was fusiform,and the matrix lamellae were arranged orderly and contained more starch grains.After low temperature stress,the starch granules of Vision decreased greatly,the size of starch granules became smaller than that of normal temperature,chloroplast membranous damage,liquid exosmosis,chloroplast matrix layer disorder and partial fracture,stomata were closed,intercellular carbon dioxide concentration increased significantly,stomatal conductance,net photosynthetic rate and transpiration rate decreased significantly.The grana of TSG10 with strong cold resistance could still maintain the integrity of its stacking structure,most of the matrix lamellae were intact and continuous,a few lamellar membranes were damaged,more starch grains were accumulated in the chloroplast,the tonoplast was intact,the stomata were semi-closed,and the increase of intercellular carbon dioxide concentration and the decrease of net photosynthetic rate were significantly lower than those of Vision.TSG10,a winter rape variety with strong cold resistance under low temperature stress,was studied by 2-DE.The results showed that 20 differentially expressed proteins were successfully identified,which were mainly involved in energy metabolism,oxidative phosphorylation,amino acid metabolism,carbon sequestration of photosynthetic organisms,scavenging of active oxygen,photosynthetic proteins,transport and catabolism.Among them,low temperature responsive proteins include S-AdenosylMethionine synthetase(spot9),phospholipid hydroglutathione peroxidase(spot11),superoxide dismutase-Fe(spot2),and photosynthesis-related proteins are β-carbonic anhydrase(spot1),chloroplast β-carbonic anhydrase(spot3),phosphoribosyl kinase(Spot 20)and transketonolase(Spot 10).Using proteomic technology,the cold-resistant winter rape variety(NS)and cold-sensitive winter rape variety(NF)were treated with-4 C for 0 h(T0),12 h(T1)and 24 h(T2).The results showed that 1978 DAP,1235 DAP and 1543 DAP were identified in NST0_NFT0,NST1_NFT1 and NST2_NFT2,respectively.A total of 34 shared DAP up-regulated in NST1_NFT1 and / or NST2_NFT2 but not expressed in NST0_NFT0 were identified.On the basis of functional analysis,we concluded that these 34 proteins are involved in the biosynthesis of phenylalanine,tyrosine and tryptophan,flavonoid biosynthesis,lysine degradation and biosynthesis of terpenoid quinones such as ubiquinone.It is involved in the formation of cold resistance of Brassica napus.Several candidate proteins(TrpD,GOT1,TAT,atoB,ALDH,CA4 H,4CL,VTE1 and FLS)are related to the synthesis of compounds and antioxidants related to cold resistance in winter rape(Brassica napus L.).