| Chinese fir(Cunninghamia lanceolata)is one of the important fastgrowing timber forest and plantation trees mainly distributed in the south of China.Continuous planting o Chinese fir plantation results in soil fertility decline and nutrient loss,at the same time,it is difficult to use the insoluble phosphorus in soil,which affects the yield and quality of forest trees.Therefore,the response mechanism of Chinese fir to low phosphorus stress has always been a hot research topic,however,the study mainly focused on the physiological and morphological changes of Chinese fir under low phosphorus stress,and the interaction between specific regulatory proteins and related proteins in Chinese fir roots under low phosphorus stress,little is known about which pathways are involved in adaptation to low-phosphorus environments.Therefore,in this study,the root tissue proteins of Chinese fir clones collected by the Chinese Fir Engineering Technology Research Center of the State Forestry and grass bureau were extracted.The root tissue proteins of Chinese fir clones with obvious growth difference under low phosphorus stress,such as No.1,No.4,No.10,No.020,No.24,No.25,No.28,No.32,No.36 and No.061,were extracted,the construction of Chinese fir antibody library was studied,and the study objects of active and passive phosphorus-tolerant type(No.25 and No.36)Chinese fir clones were selected,two different phosphorus levels and different time gradients(0h,1 h,3 h,9 h,27 h,81 h and 243 h)were used to screen the different proteins of different phosphorus-tolerant clones of Chinese fir,and to analyze the related protein pathways,it is important to construct the interaction network of proteins for further elucidating the molecular regulation mechanism of Chinese fir under low phosphorus stress,which can solve the problem of phosphorus deficiency from Chinese fir itself.The main results were as follows:(1)Under low phosphorus stress,the total gray values of protein samples from the roots of different Chinese fir clones were basically the same,and without high abundance.After successfully extracting the denatured protein from mixed sample of Cunninghamia Lanceolata,the mixed sample extracts were injected into M1,M2 and M3 mice for Elisa detection,it was found that the Elisa titer of M3 in diluted 1K,2K,5K,10 K,20K,40 K and 80 K of mice was higher than 0.5,which was significantly better than that of the other two mice,therefore,mouse M3 was selected for the next step of antibody chip preparation.(2)More than 10,000 monoclonal antibody were made into antibody chip,and the chip scanning showed that the sample was bright,uniform in size and orderly arranged,with less than 5 % of connecting points and leakage points.The fluorescence signals of the two adjacent chips correspond to the same region,and the repeatability is high.The internal reference point BSA value is normal,the signal intensity is gradient distribution,the signal-to-noise ratio(fluorescence signal value F /background noise signal value M)>10,the basic conditions of point chip making are satisfied.The results of density plot show that the distribution of the main peak is about 9,and the distribution of the data is consistent after the homogenization treatment,which indicates the success of the preparation of antibody chip.(3)The different protein of Chinese fir clones with different phosphorus levels were screened by antibody chip.It was found that the protein expression level of NO.36 was generally higher in the condition of phosphorus supply than that in the condition of phosphorus supply,and the change trend of protein expression level of No.36 was first increased and then tended to be stable or slightly decreased in the condition of phosphorus supply,but not phosphorus supply,its early change is more smooth rise,after reaching the inflection point,then increase or decrease significantly.For No.25,the change tendency of the treatments with and without phosphorus supply was larger than that of No.36,and the value of the treatments without phosphorus supply generally increased first and then decreased or tended to be stable.In addition,under the condition of phosphorus supply,No.36 is generally higher than No.25,and the overall variation of No.36 is more stable than No.25.In the environment without phosphorus supply,the change trend of the clone of No.36 was more gentle than that of No.25.(4)In this study,the first batch of 680 candidate antibodies selected from the results of chip screening were tested by WB,73 different antibodies were obtained,and 73 different antibodies were identified by protein pair analysis.It was found that the gray values of these 73 protein pairs were significantly different in different phosphorus supply levels or in different Chinese fir clones.Some of the proteins were selected at random to carry out IP,then silver-stained the products,and the special bands with the same size as the different WB were observed in the IP channels.(5)Protein FLX-like 3,enolase and glutamine synthetase were selected randomly after mass spectrometry to study protein-protein interaction.The unique peptide 2 protein was identified as the interacting protein,and 39 proteins interacting with the Protein FLX-like 3,60 proteins interacting with the protein enolase and 55 proteins interacting with the protein glutamine synthetase were found.Go analysis of Protein FLX-like 3,enolase and glutamine synthetase,it was found that the three proteins and other proteins constitute dozens of functional pathways,including response to cadmium ion,glycolysis and Lignin biosynthesis,in addition,there were more than 6 proteins responding to Cadmium Ions and 4 proteins responding to cadmium ions in the process of Glycolysis when the three proteins interacted with other proteins.The KEGG pathway enrichment analysis of 39 proteins interacting with Protein FLXlike 3,60 proteins interacting with enolase and 55 proteins interacting with glutamine synthetase was performed,it was found that the differentially expressed proteins were mainly concentrated in the pathway of Amino acid biosynthesis,glycolysis and glycolysis,carbohydrate regeneration,carbon metabolism and other organic acid metabolism.Through the establishment of the network interaction map of the proteins obtained by mass spectrometry analysis,it was found that most of the identified proteins had interactions with three randomly selected target proteins,the specific function needs to be further verified by Arabidopsis thaliana.(6)Under low phosphorus stress,the contents of MDA,SOD and POD in the roots of different Chinese fir clones increased at first and then decreased with the increase of time,and the activities of three enzymes in No.25 Chinese fir were higher than those in No.36 Chinese fir.With the increase of stress time,the activities of three enzymes of No.36 Chinese fir were higher than that of No.25.The results also showed that the response of No.25 to low phosphorus stress was faster than that of No.36,which showed active phosphorus seeking.(7)In conclusions,we constructed the first monoclonal antibody library targeting the Chinese fir proteome,and constructed an antibody library of 10,368 monoclonal antibody,covering about 2,488 proteins.The protein(group)of different phosphorus-tolerant clones of Chinese fir were screened by antibody chip technology,and 680 differences were found.After identification,73 differential proteins were identified,including MTAN involved in DNA methylation,SAMS involved in ethylene biosynthesis,FLX-like3,5’-methylthioadenosine/Sadenosylhomocysteine nucleosidase、S-adenosylmethionine synthase 2;L-Asparagine is involved in the synthesis of glutamine synthetase,a protein called jacalin-related lectin that regulates the correct aggregation of BGLU 23/PYK 10 during tissue injury,and Catalase,which protects cells from the toxic effects of hydrogen peroxide,enolase,a protein that catalyzes the formation of flavonols from dihydroflavonols.It is suggested that further studies should be carried out on the functions of the related proteins and the functions of the pathways involved,in order to improve the yield of Chinese fir in the red soil region of south China,a model was established in which a specific substance or several substances together regulated the responses of Chinese fir to low phosphorus stress. |
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