Study On Differentially Expressed Proteome In Response To Low Temperature In Alfalfa

Alfalfa, known as the "king of the grazing ", is an important perennial forage grass with its rich nutrition, good palatability, high yield and strong resistance, and is one of the main varieties of forage grass planted in north region of China. However, production,quality and species diversity of alfalfa has usually been limited by low temperature in the north area, especially in Hei Longjiang province. The research on cold stress response mechanism of alfalfa has certain theoretical significance and practical value.In this study, two cultivars, Zhaodong alfalfa and WL525 HQ, with different cold hardiness were chosen as experiment material through field experiments. The method of low-abundance proteins extraction was established to improve the detection of proteins by two-dimensional electrophoresis(2-DE). The physiological results and differential proteins were analyzed by biochemical technology and 2-DE combined with mass spectrometry, respectively, RT-PCR was used to detect the results of proteins changes,to analyze the regulatory mechanism in response to low temperature on level of physiology and proteomics. The results of the study were as follows:1. The field text showed there was no difference on growth period and agronomy traits among Zhaodong alfalfa, WL323 HQ, Sanditi, Victoria and WL525 HQ. However,significant difference of the regeneration height after mowing and grass reviving rate were obvious. Cold resistance of five cultivars was Zhaodong alfalfa > WL323 HQ >Sanditi ≥ Victoria > WL525 HQ. And there was significant difference of cold resistance ability between Zhaodong alfalfa and WL525 HQ.2. The results of physiological experiment showed the physiological parameters were changeful in two cultivars alfalfa under cold stress. The content of chlorophyll reduced under cold stress and the reducing rate in WL525 HQ was larger than in Zhaodong alfalfa. The activity of POD, APX, GR, DHAR, MDHAR and GSH-PX increased, and of SOD increased at first and then decreased. Except APX, the growth of antioxidase in Zhaodong alfalfa was larger than in WL525 HQ, which showed positive correlation with cold resistance ability. The relative conductivity increased under cold stress, and the results showed larger increasing ratio in WL525 HQ. The accumulation ofsoluble sugar was found under cold stress, and the larger accumulation was observed in Zhaodong alfalfa. According to determination of free amino acid content, 14 kinds of free amino acids were detected in alfalfa leaf(methionine and glycine be not detected).The content of 14 kinds of free amino acid changed under cold stress, and changing of11 kinds were observed in WL525HQ(alanine, leucine, phenylalanine unchanged).Results showed the pattern of diversity and differences between the varieties during cold stress. Free proline content in Zhaodong alfalfa was significantly higher than in WL525 HQ, showed a positive correlation with its cold resistance ability.3. Protein extraction solution of Mg/NP-40 combined with different concentrations of PEG was applied to screening the suited method to extract the low-abundance proteins in alfalfa. The results show that the concentration of 17.5% PEG is the most suitable for the extraction of low abundance proteins, which could remove most RuBisCO and improve the resolution ratio of proteins.4. Holoproteins and low-abundance proteins of alfalfa leaf were extracted,respectively, and 67 differential proteins in response to low temperature, changes of the relative abundance over 1.5 fold, were identified by two-dimensional gel electrophoresis and mass spectrometry technology. Based on the protein function, 67 identified proteins were classified into six categories: photosynthesis related proteins(42%), energy metabolism related proteins(18%), stress response and redox protein related proteins(13%), biosynthesis related proteins(13%), protein processing related proteins(9%) and unknown function proteins(5%).5. In our study, the relative abundance of 49 proteins in Zhaodong alfalfa changed in response to cold stress, of which 15 proteins also involved in the low temperature response in WL525 HQ with different changing pattern. The relative abundance of the remaining 34 proteins only changed in Zhaodong alfalfa with the constant expression quantity in WL525 HQ. The relative abundance of 47 proteins changed over 1.5 fold in the early cold stress(12 h), and 20 proteins expressed differently when exposed to cold stress for 7 d. More than half of the proteins maintain the same expression quantity as12 h in Zhaodong alfalfa at 7 d.6. Under cold stress, the relative abundance of 24 proteins changed to response low temperature in WL525 HQ, of which 9 proteins only differently expressed in WL525 HQ.Additionally, 9 proteins didn’t change during cold stress, but maintained the highrelative abundance. The relative abundance of 20 proteins changed over 1.5 fold in the early cold stress(12 h), and 22 proteins expressed differently when exposed to cold stress for 7 d.7. Different proteins were validated in mRNA and protein levels of the alfalfa under cold stress for 0, 12 h and 7 d. Different proteins were involved in photosynthesis,energy metabolism, stress and redox, biosynthesis, protein processing and other functions. The results were consistent with mass spectrometry at the ratio of 50% and66.7% in Zhaodong alfalfa and WL525 HQ, respectively. The relative abundance changing of RuBisCO large subunit, aconitate hydratase and eukaryotic translation initiation factor were highly linear correlated to gene changes in Zhaodong alfalfa. And there was highly linear correlation between protein level and gene level of RuBisCO large subunit, monodehydroascorbate reductase, cinnamoyl-CoA reductase and eukaryotic translation initiation factor in WL525 HQ.